Agents for treatment of HCV and methods of use

ABSTRACT

An amphipathic helix at the approximate N-terminus of Hepatitis C virus (HCV) nonstructural proteins mediates the association of these proteins with cytoplasmic membranes in infected cells. This association is essential for replication. Thus, assessing the ability of compounds or protocols to disrupt the association of such helices with cytoplasmic membranes permits identification of compounds and protocols which are useful in the treatment of HCV infection. Also useful in the invention are mimics, or function-disrupting ligands, of an amphipathic helix of the nonstructural proteins described herein and antibodies and fragments thereof immunoreactive with said helix.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.10/481,261, filed Jul. 30, 2004 (now U.S. Pat. No. 7,326,536, issuedFeb. 5, 2008), which is a U.S. national phase filing under 35 U.S.C.§371 of International Patent Application No. PCT/US02/13951, filed May3, 2002, which claims the benefit of U.S. Provisional Patent ApplicationNo. 60/288,687, filed May 3, 2001 and U.S. Provisional PatentApplication No. 60/316,805, filed Aug. 31, 2001, which applications areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention relates generally to Hepatitis C virus (HCV) infection,and more specifically to interrupting the mechanism of infection by HCVand to methods to identify agents, which effect this interruption. Theinvention also relates to interfering with the ability of HCV componentsto bind with cellular membranes of an infected cell.

BACKGROUND

Hepatitis C virus (HCV) establishes a chronic infection in a highpercentage of infected individuals and is associated with progressiveliver pathology, including cirrhosis and hepatocellular carcinoma.Antiviral drugs such as interferon alpha and ribavarin have had limitedsuccess in controlling HCV infection. As a result, it has become theleading cause for liver transplantation in the US. The HCV polyproteincomprises, from the amino terminus to the carboxy terminus, the coreprotein (C), the envelope proteins (E1 and E2), p7, a membrane boundprotein, whose function is unknown and the non-structural proteins (NS2,NS3, NS4A, NS4B, NS5A and NS5B) which are believed to be important forreplication. C codes for the core nucleocapsid protein, E1 and E2 areenvelope proteins that coat the virus, NS2, NS3 and NS4A are involved inproteolytic processing of the HCV polyprotein, and NS5B has RNApolymerase activity. The functions of NS4B and NS5A are unknown.

Hepatitis C virus is a significant cause of morbidity and mortality,infecting over 100,000,000 people worldwide. Annual HCV related costs inthe United States are about $1 billion. Current therapies are clearlyinadequate; the best available treatment at present is the combinationof interferon and ribavirin, a treatment which is inconveniently lengthyas it typically lasts over one and a half years, difficult to toleratein that most patients have flu-like symptoms, and extremely expensive asthe cost is in the range of thousands of dollars annually. Not only doesthe present treatment have these disadvantages, but it is also notparticularly effective.

Certain interactions of viral proteins with cell membranes havepreviously been described. For example, in poliovirus and Hepatitis Avirus, the nonstructural protein 2C contains a membrane associatingamphipathic helix (See Teterina, N. L., et al., J. Virol. (1997)71:8962-8972 (poliovirus); and Kusov, Y. Y., et al., Arch. Virol. (1998)143:931-944 (Hepatitis A). This membrane association appears to play arole in RNA synthesis in poliovirus (Paul, A. V., et al., Virol. (1994)199:188-199). Replication complexes are localized on the hostendoplasmic reticulum (ER) and Golgi in the case of poliovirus (Bienz,K., et al., J. Virol. (1992) 66:2740-2747), and infection withpoliovirus induces rearrangements of membranes derived from host ER andGolgi (Schlegel, A., et al., J. Virol. (1996) 70:6576-6588).

It is also known that the Hepatitis C nonstructural 5A proteinillustrated below is a potent transcriptional activator (Kato, N., etal., J. Virol. (1997) 71:8856-8859); that amino terminal deletionmutants of Hepatitis C virus nonstructural protein NS5A function astranscriptional activators in yeast (Tanimoto, A., et al., Biochem.Biophys. Res. Commun. (1997) 236:360-364); and that this nonstructuralprotein physically associates with p53 and regulates p21/Waf1 geneexpression in a p53 dependent manner Majumder, M., et al., J. Virol.(2001) 75:1401-1407).

It has also been reported that a number of positive strand RNA viruses,like HCV, replicate in association with cytoplasmic membranes, althoughthe precise manner of such association and replication is notunderstood. See, for example, Lazarus, L. H., et al., J. Gen. Virol.(1974) 23:213-218 (foot and mouth disease); Bienz, K., et al., Virol.(1980) 100:390-399 (polio); Froshauer, S., et al., J. Cell. Biol. (1988)107:2075-2086 (alphavirus); Chu, P. W., et al., Arch. Virol. (1992)125:177-191 (Kunjin virus); Rice, C. M., in Fields Virology, Fields, B.N., et al., Ed. (1996) Lippincott-Raven Publications: Philadelphia, Pa.,pages 931-959 (Flaviviridae).

It is also known that NS5A, the nonstructural Hepatitis C protein usedfor illustration below is associated with cell membranes (Selby, M. J.,et al., J. Gen. Virol. (1993) 74:1103-1113; Hijikata, M., et al., Proc.Natl. Acad. Sci. USA (1993) 90:10773-10777; Moradpour, D., et al.,Hepatol. (1998) 28:192-201). This protein, NS5A, also has been reportedto interact with other host cell proteins such as PKR protein kinase(Gale, M. J. J., et al., Virol. (1997) 290:217-227) and a SNARE-likeprotein (Tu, H., et al., Virol. (1999) 263:30-41). NS5A has also beenimplicated in determining the response to interferon therapy in somegroups of patients (Gale, M. J. J., et al., Virol., supra); Enomoto, H.,et al., N. Engl. J. Med. (1996) 334:77-81.

There exists a need in the art for compositions, including peptidetherapeutics, and methods employing the same, to prevent or inhibitinfections due to Hepatitis C Virus, including inhibiting replicationand/or pathogenesis due to HCV, with minimal or no adverse side effects.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that the presence of anamphipathic helix located near the N-terminus of certain nonstructuralproteins of HCV is required to associate these proteins with thecellular membranes in the infected host. Three such HCV nonstructuralproteins, NS4B, NS5A and NS5B are known to be associated with the hostcellular membranes and thus participate in the replication of HCV, whichis considered to occur in association with cytoplasmic membranes andthus are implicated in the participation of replication of HCV. Theseproteins contain at least one amphipathic helix of about 20-25 aminoacids in the N-terminal region which, when disrupted, can result infailure of these proteins to properly associate with cellular membranes.The ability of candidate reagents to interfere with this binding canreadily be assessed by linking the amphipathic helix to a label, such asa reporter protein, and assessing the effects of candidate agents on theposition of the reporter in the cell. Such an assay can be conducted incell culture, and thus can overcome one significant obstacle todiscovery of additional treatments for Hepatitis C viral infection.

There is a plethora of alternative methods other than thisstraightforward approach to assess the ability of a candidate substanceor protocol to disrupt the necessary binding between the relevantamphipathic helix and the cytoplasmic cellular membranes. Theseapproaches will be described in greater detail below.

Thus, in one aspect, the invention is directed to a method to identify acompound or protocol which is useful in treating HCV infection, whichmethod comprises assessing the ability of a candidate compound orprotocol to disrupt the binding of a peptide comprising an amphipathichelix derived from a Hepatitis C nonstructural protein, in particularNS4B, NS5A or NS5B, to cellular membranes.

This can be done, for example, by simply assessing the binding of acandidate compound to said peptide, wherein a compound which binds saidpeptide is identified as an anti-HCV agent. Alternatively, the abilityof a candidate compound to inhibit the binding of a peptide containingthe amphipathic helix to a membrane preparation from eukaryotic cellscan be evaluated. In these aspects, the assay can be carried out usingstraightforward laboratory procedures, not even involving cell culture.

Another alternative method to identify compounds that are effectiveanti-HCV agents comprises providing cells which contain an amphipathichelix derived from Hepatitis C viral nonstructural proteins NS4B, NS5Aor NS5B coupled to a detectable label; treating one sample of said cellswith the candidate compound or protocol and maintaining a sample of saidcells in the absence of said candidate compound or protocol; comparingthe location of the label in cells treated with said compound orprotocol as compared to cells not treated with said compound orprotocol, whereby when cells in the presence of the candidate compoundor protocol show a different cellular distribution of label as comparedto cells not treated with said compound or protocol, said compound orprotocol is identified as an agent or protocol for treating HCV.

Alternatively, a compound or protocol can be identified as an anti-HCVagent by linking the amphipathic helix derived from the N-terminalregion of certain HCV nonstructural proteins and a reporter or label andobserving the behavior of the reporter intracellularly. This can be donedirectly as described above by observing the intracellular location of areporter that is a simple label or can take advantage of secondaryeffects of the reporter. For example, as described below, disrupting thebinding of the nonstructural protein NS5A with the cytoplasmic membraneby disrupting the amphipathic helix results in translocation of thenonstructural protein to the nucleus. Thus, if the reporter includes anuclear localization signal, the disruption of interaction with thecytoplasmic membrane can be measured indirectly by assessing the effectsof translocation of the helix and its reporter functions to the nucleusas will be described in further detail below.

In another aspect, the invention is directed to methods of treating HCVby administering to a subject in need of such treatment an agent orprotocol identified by the methods described above or by an agent orprotocol which disrupts the interaction of an amphipathic helix derivedfrom HCV nonstructural proteins with cellular membranes or with an agentthat binds to said helix. The invention is also directed to agents andprotocols identified by the method of the invention. The nature of someof the agents and protocols which will disrupt the binding of thesehelices to cellular membranes is apparent without the necessity forcarrying out the above-described assays. For example, one approach is toemploy the amphipathic helix itself as a competitor for the helix as itexists in the infective agent. Thus, peptides having the amino acidsequence corresponding to the sequence in the helices of the various HCVnonstructural proteins can be used as pharmaceuticals to compete withthe helices contained in the nonstructural proteins themselves. Inaddition, molecules which mimic the pattern of hydrophilic orhydrophobic faces of the helix can be employed. Peptides which containnon-amide bonds but which retain the same essential backbone shape couldalso be used, as could aptamers which assemble to obtain the essentialfeatures of the helix. It may be possible by ascertaining thethree-dimensional structure of the helix, the appropriate antibodies, oraptamers to design rationally organic molecules that assume the samecharge/spatial configuration. Thus, the invention also includes theseembodiments.

In another aspect, the invention is directed to a peptide of not morethan about 60 amino acid residues, which is an HCV nonstructural proteinamphipathic helix, where the peptide optionally contains non-peptideisosteric linkages, and where the peptide is optionally coupled to anadditional heterologous second peptide or to an additional component.

In yet another aspect, the invention provides an isolated peptide thatinterferes with the binding of an amphipathic helix derived from HCVnonstructural proteins with cellular membranes.

In still another aspect, the invention provides a peptide that inhibitsthe association of an amphipathic helix present in the N-terminus of anHCV nonstructural protein with cytoplasmic membranes of a cell andhaving an amino acid sequence SGSWLRDVWDWICTVLTDFKTWLQSKL (SEQ ID NO:14), and variants and mutants thereof, and wherein the peptide isidentified by assessing the ability of the peptide to interfere with thebinding of an amphipathic helix present in the N-terminus of an HCVnonstructural protein with cytoplasmic membranes of a eukaryotic cell,where a peptide which interferes with binding is identified as useful intreating HCV infection.

In yet another aspect, the invention provides a method of screening fora peptide useful as an inhibitor of the binding of an HCV nonstructuralprotein with cytoplasmic membranes of a eukaryotic cell by contacting anamphipathic helix present in the N-terminus of an HCV nonstructuralprotein with cytoplasmic membranes of a eukaryotic cell, both in thepresence and absence of a test compound, wherein a decreased level ofbinding in the presence of the compound as compared to the level in theabsence is indicative of a peptide inhibitor.

In another embodiment, the invention provides an isolated peptideselected from complementary peptides or competitive inhibitors,functional fragments, mutants or variants thereof, wherein the isolatedpeptide causes inhibition of infection, replication, or pathogenesis ofHepatitis C Virus in vitro or in vivo when introduced into a host cellcontaining said virus, and wherein said isolated peptide exhibits anIC₅₀ in the range of from about 0.0001 nM to about 100 μM in an in vitroassay for at least one step in infection, replication, or pathogenesisof said virus.

In yet another embodiment, the invention provides a composition of oneor more isolated peptides of the invention, functional fragments,mutants or variants thereof, and a carrier, a diluent, an excipient or abuffer.

In still another embodiment, the invention provides a pharmaceuticalcomposition of one or more isolated peptides of the invention,functional fragments, mutants or variants thereof, and apharmaceutically acceptable carrier, diluent, excipient, or buffer.

In another embodiment, the invention provides a method of preventing ortreating HCV infection in a patient in need thereof, comprisingadministering to said patient an anti-HCV effective amount of anisolated peptide of the invention, functional fragments, mutants orvariants thereof.

In another embodiment, the invention provides use of an isolated peptideselected from the peptide of the invention, functional fragments,mutants or variants thereof in the preparation of a medicament for theprevention or treatment of HCV infection in a human patient in needthereof.

In yet another embodiment, the invention provides a method of treatmentof HCV infection in a patient in need thereof, comprising administeringto said patient an anti-HCV effective amount of an isolated peptide ofthe invention, comprising the NS5A amphipathic helix or a functionalfragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequences (SEQ ID NOS: 1-13) which compriseamphipathic helices in the N-terminal region of NS4B, NS5A and NS5B ofHepatitis C virus genotype 1a. Helices of NS5A from a variety ofisolates are particularly exemplified.

FIGS. 2A-2J show the helical conformation of sequences in the variousisolates of NX5A (SEQ ID NOS: 469 and 5-13, respectively).

FIGS. 3A-3C, lower panels, show the conformation of amino acid sequencesin positions 4-27 of NS5A which form the N-terminal helix in the nativeprotein (FIG. 3A; SEQ ID NO: 297) but wherein the helical structure isdeleted (FIG. 3B; SEQ ID NO: 297) or wherein the hydrophobic region isinterrupted (FIG. 3C; SEQ ID NO: 470).

FIGS. 3A-3C, upper two panels, show the effect of the alterations to thehelical structure on the cellular distribution pattern of NS5A. FIG. 3Ashows the pattern obtained when the helix is not disrupted; FIGS. 3B and3C show the patterns obtained when the helix is deleted or thehydrophobic face is disrupted.

FIG. 4A-4C show the distribution patterns of the N-terminal amphipathichelix of NS5A coupled to green fluorescent protein (GFP) in comparisonto suitable controls. FIG. 4B shows the localization pattern of thefusion protein of the helix with GFP; FIG. 4A shows the pattern for GFPalone, and FIG. 4C shows the pattern obtained when the hydrophobic faceof the helix is disrupted.

FIGS. 5A and 5B, upper panels, show schematics of subgenomic repliconsof HCV used in HCV replication assays; FIGS. 5A and 5B, lower panels,show the results of these assays.

FIGS. 6A-6C show the results of a membrane floatation assay for bindingof the NS5A amphipathic helix with microsomal membrane.

FIGS. 7A and 7B show the effects of a peptide useful as an anti-HCVagent on binding of NS5A to the microsomal membrane using a floatationassay.

FIG. 8 shows pharmacologic inhibition of NS5A membrane association.

DETAILED DESCRIPTION OF THE INVENTION

The Hepatitis C virus has been well studied and the genome has beencompletely sequenced. The genome of HCV is a single-stranded RNA of 9.6kb that encodes a single approximately 3,010 amino acid polyprotein.This polyprotein is proteolytically processed into structural proteinsand nonstructural proteins. HCV, like other positive strand RNA viruses,is thought to replicate in association with cytoplasmic membranes; andthe approximately 447 amino acid nonstructural protein, NS5A is known toassociate with host cell membranes. This protein is thus believed toplay a key role in replication. The discovery of alternative treatmentshas been hampered by the lack of a cell culture model for HCV. Thepresent invention overcomes this problem, and provides a convenientassay for identifying agents that can directly and specifically curtailthe course of HCV infection. As noted above, the present inventionresides in the discovery of a mechanism to disrupt association betweencertain nonstructural proteins of the virus and cytoplasmic membranes.

Administration of peptides and proteins intracellularly has been limitedby the lack of consistent and efficient uptake across the cell membrane.However, various membrane translocation sequences (MTS) have beenidentified which allow the transduction of peptides across the plasmamembrane (for example, References 10, 13, 14, 16, 17, 20, 21 and 22).While unknown, it is hypothesized that the mechanism of the transductionis that an MTS is able to transport both peptides and full-lengthproteins into the cytoplasm of cells. It is believed that the separatepeptides form a noncovalent complex in solution that is then transportedinto the cell.

It has now been found that an amphipathic helix consisting of about20-25 amino acids is critical to the association of NS5A withcytoplasmic membranes, for example endoplasmic reticulum (ER), as aresimilar helices found in NS4B and NS5B. As shown below, when theamphipathic nature of the helix is disrupted, NS5A no longer associateswith cell membranes; in addition, the helix when coupled to othersubstances, including other proteins, effects association of the carriedcomponent to the cell's cytoplasmic membranes. As used herein,“cytoplasmic membrane” refers to a structure contained in the cellularcytoplasm, which can generally be recognized as a membrane structure forexample, membranes of the endoplasmic reticulum (ER). The membranes mayinclude protein receptors or other elements in addition to the morehydrophobic portions of the membrane per se which may account at leastin part for the association of the amphipathic helix to the “cytoplasmicmembrane”. No theory is advanced as to the mechanism whereby theamphipathic helix associates itself with the cytoplasmic membrane, andthus the precise elements contained in the membrane, which areresponsible for this association, are not defined. The observationsrelated to the screening methods described below are not dependent onany definition: intracellular embodiments of these assays rely on directobservation of the cellular distribution of the helix and its label andstudies which involve membrane preparations ex vivo employ compositionsof cytoplasmic membranes as conventionally prepared, without regard tosubstructure. This amphipathic helix feature is found in nonstructuralproteins of isolates of many strains of HCV of various geographicalorigins. It is also demonstrated below that disruption of theamphipathic nature of the helix of NS5A prevents replication of HCV RNA.

FIG. 1 shows the amino acid sequences of the amphipathic helices thathave been found near to the N-terminus of three nonstructural proteinsfrom prototype genotype 1a representing amino acids 7-34 of NS4B (SEQ IDNO: 1), amino acids 5-26 of NS5A (SEQ ID NO: 2), and two amphipathichelical regions of NS5B, amino acids 65-87 (SEQ ID NO: 3) and 107-125(SEQ ID NO: 4). This figure also shows the relevant sequences in NS5Afor additional genotypes 1b (acc. No. P26663) (SEQ ID NO: 5); 2a (acc.No. P26660) (SEQ ID NO: 6); 2b (acc. No. AB030907) (SEQ ID NO: 7); 3a-K(acc. No. D28917) (SEQ ID NO: 8); 3a-NZL (acc. No. D17763) (SEQ ID NO:9); 3b (acc. No. D49374) (SEQ ID NO: 10); 4a (acc. No. Y11604) (SEQ IDNO: 11); 10a (acc. No. D63821) (SEQ ID NO: 12); and 11a (acc. No.D63822) (SEQ ID NO: 13). The sequences listed in FIG. 1 are not anexhaustive list of the amino acid sequences of amphipathic helices ofthese three nonstructural proteins. NS4B and NS5B may be expandedsimilarly to NS4A in FIG. 1. Although the sequences per se are notcompletely homologous, they are all capable of forming amphipathichelices. (See FIGS. 2A-2J which show these helices from the NS5Aproteins.) Thus, it is clear that the presence of the N-terminalamphipathic helix, containing a sequence of approximately 20-25 aminoacids is widespread in the nonstructural proteins of HCV and in variousisolates.

As used herein, the term “HCV nonstructural protein amphipathic helix”refers to a sequence of at least 15 amino acids, preferably at least 20amino acids and preferably not more than 30, more preferably not morethan 25 amino acids which has the amino acid sequence selected from thegroup consisting of those set forth in FIG. 1, or which is at least 80%homologous, preferably at least 90% homologous and more preferably atleast 95% homologous to at least one of said sequences and which retainsthe ability to form an amphipathic helix. While it is known that certainamino acid substitutions will result in peptides, which, while theyretain the required degree of homology (sequence identity), will disruptthe formation of the helix, the nature of these substitutions is alreadyunderstood by those of ordinary skill and can be avoided, orpurposefully used, as desired. Insertion of, for example, disruptiveproline residues, is understood to be undesirable. Thus, it is wellwithin ordinary skill to substitute one or more amino acids in thesesequences to obtain substitute helices with the required degree orhomology, avoiding unsuccessful substitutions.

It should be noted that in certain embodiments of the invention, theamphipathic helix may be other than a peptide; for example, it mayconstitute a pseudopeptide where the native peptide linkages aresubstituted by isosteres as is understood in the art. Similarly,alternative polymeric structures can be designed which mimic the chargeand shape distribution of these amphipathic helices and mutants andvariants thereof. These homologs and analogs are included within thescope of the invention and are useful both in therapeutic protocols andin assay systems for ascertaining alternative compounds and protocolswhich will be useful in treating HCV infection.

As used herein, the term “amino acid” is applicable not only to cellmembrane-permeant peptides, but also to amphipathic helices and anti-HCVpeptides useful as pharmaceutical agents, i.e., all the individualcomponents of the present compositions.

The term “amino acid” is used herein in its broadest sense, and includesnaturally occurring amino acids as well as non-naturally occurring aminoacids, including amino acid analogs and derivatives. The latter includesmolecules containing an amino acid moiety. One skilled in the art willrecognize, in view of this broad definition, that reference herein to anamino acid includes, for example, naturally occurring proteogenicL-amino acids; D-amino acids; chemically modified amino acids such asamino acid analogs and derivatives; naturally occurring non-proteogenicamino acids such as norleucine, β-alanine, ornithine, etc.; andchemically synthesized compounds having properties known in the art tobe characteristic of amino acids. As used herein, the term “proteogenic”indicates that the amino acid can be incorporated into a peptide,polypeptide, or protein in a cell through a metabolic pathway.

The incorporation of non-natural amino acids, including syntheticnon-native amino acids, substituted amino acids, or one or more D-aminoacids into the present anti-HCV peptides of the present invention isadvantageous in a number of different ways. D-amino acid-containingpeptides exhibit increased stability in vitro or in vivo compared toL-amino acid-containing counterparts. Thus, the construction of peptidesincorporating D-amino acids can be particularly useful when greaterintracellular stability is desired or required. More specifically,D-peptides, etc., are resistant to endogenous peptidases and proteases,thereby providing improved bioavailability of the molecule, andprolonged lifetimes in vivo when such properties are desirable. When itis desirable to allow the peptide, etc., to remain active for only ashort period of time, the use of L-amino acids therein will permitendogenous peptidases, proteases, etc., in a cell to digest the moleculein vivo, thereby limiting the cell's exposure to the molecule.Additionally, D-peptides, etc., cannot be processed efficiently formajor histocompatibility complex class II-restricted presentation to Thelper cells, and are therefore less likely to induce humoral immuneresponses in the whole organism.

One factor that can be considered in making such changes is thehydropathic index of amino acids. The importance of the hydropathicamino acid index in conferring interactive biological function on aprotein has been discussed by Kyte and Doolittle (1982, J. Mol. Biol.,157: 105-132). It is accepted that the relative hydropathic character ofamino acids contributes to the secondary structure of the resultantprotein. This, in turn, affects the interaction of the protein withmolecules such as enzymes, substrates, receptors, DNA, antibodies,antigens, etc.

Based on its hydrophobicity and charge characteristics, each amino acidhas been assigned a hydropathic index as follows: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine(+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine(−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline(−1.6); histidine (−3.2); glutamate/glutamine/aspartate/asparagine(−3.5); lysine (−3.9); and arginine (−4.5).

As is known in the art, certain amino acids in a peptide, polypeptide,or protein can be substituted for other amino acids having a similarhydropathic index or score and produce a resultant peptide, etc., havingsimilar biological activity, i.e., which still retains biologicalfunctionality. In making such changes, it is preferable that amino acidshaving hydropathic indices within ±2 are substituted for one another.More preferred substitutions are those wherein the amino acids havehydropathic indices within ±1. Most preferred substitutions are thosewherein the amino acids have hydropathic indices within ±0.5.

Like amino acids can also be substituted on the basis of hydrophilicity.U.S. Pat. No. 4,554,101, herein incorporated by reference, disclosesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein. The following hydrophilicity valueshave been assigned to amino acids: arginine/lysine (+3.0);aspartate/glutamate (+3.0±1); serine (+0.3); asparagine/glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5±1);alanine/histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine/isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); and tryptophan (−3.4). Thus, one amino acid in a peptide,polypeptide, or protein can be substituted by another amino acid havinga similar hydrophilicity score and still produce a resultant peptide,etc., having similar biological activity, i.e., still retaining correctbiological function. In making such changes, amino acids havinghydropathic indices within ±2 are preferably substituted for oneanother, those within ±1 are more preferred, and those within ±0.5 aremost preferred.

As outlined above, amino acid substitutions in the anti-HCV peptides ofthe present invention can be based on the relative similarity of theamino acid side-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, etc. Exemplary substitutions that takevarious of the foregoing characteristics into consideration in order toproduce conservative amino acid changes resulting in silent changeswithin the present peptides, etc., can be selected from other members ofthe class to which the naturally occurring amino acid belongs. Aminoacids can be divided into the following four groups: (1) acidic aminoacids; (2) basic amino acids; (3) neutral polar amino acids; and (4)neutral non-polar amino acids. Representative amino acids within thesevarious groups include, but are not limited to: (1) acidic (negativelycharged) amino acids such as aspartic acid and glutamic acid; (2) basic(positively charged) amino acids such as arginine, histidine, andlysine; (3) neutral polar amino acids such as glycine, serine,threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and(4) neutral non-polar amino acids such as alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan, and methionine.

It should be noted that changes which are not expected to beadvantageous can also be useful if these result in the production offunctional sequences. Since small peptides, etc., can be easily producedby conventional solid phase synthetic techniques, the present inventionincludes peptides, etc., such as those discussed herein, containing theamino acid modifications discussed above, alone or in variouscombinations. To the extent that such modifications can be made whilesubstantially retaining the activity of the peptide, they are includedwithin the scope of the present invention. The utility of such modifiedpeptides, etc., can be determined without undue experimentation by, forexample, the methods described herein.

Based on the present discoveries, various methods to identify agents andprotocols which would be effective in treating HCV are made possible. Intheir simplest form, since binding between the helix and the cytoplasmicmembrane is a necessary condition for association of the nonstructuralprotein with the membrane and is required for the virus to replicate,compounds which simply bind the helix compete with the cellularmembranes for this association. When the helix is contained in asubstantially larger molecule, such as existing in the context of asignificantly longer amino acid sequence, it is possible that theremaining portion of the molecule or amino acid sequence may itselfcontain elements that are capable of affecting binding. Thus, in theassays of the invention, appropriate controls may be required to ensurethat it is the interaction of the helix with the compounds tested or themembranes tested that is being measured. Compounds that bind to thehelix would inhibit the required cellular binding, and by identifyingcompounds that bind with high affinity to the helix compounds that wouldbe successful in interfering with the binding will be found. By “highaffinity” is meant binding with a dissociation constant of <10⁻⁴,preferably <10⁻⁶, and more preferably <10⁻⁸. Dissociation constants canbe determined using methods generally known in the art; for example thehelix could be bound to solid support and labeled compound at varyingconcentrations supplied wherein the amount of label coupled to solidsupport can be readily determined.

Alternatively, the peptide comprising the helix can be displayed usingstandard phage display techniques and the phage tested for ability tobind to compounds that are coupled to solid supports. Phage display isalso utilized to screen random peptides to screen for competitiveinhibitors of the amphipathic helix. Such peptides would block virusfrom binding to the receptor.

There are a variety of techniques well known in the art for assessingbinding of compounds to targets. A wide variety of detectable labels canbe used, and the assays can be conducted in heterogeneous or homogeneousformats. For example, the compound itself can be labeled for detectionof its ability to transport label to solid support, to which solidsupport has been bound the amphipathic helix or, vice versa, theamphipathic helix can be labeled and the compound to be tested can becoupled to solid support. The assay can also be conducted in ahomogeneous format using fluorescence techniques, for example, where thefluorescence of a fluorescent label attached to the compound is alteredby virtue of its binding to a peptide the size of the amphipathic helixor larger.

The amphipathic helix itself can be supplied in the context of thenonstructural protein or fragment thereof or can be supplied per se oras a fusion protein that contains a label, such as green fluorescentprotein.

In a variation of this extracellular format, the effect of individualcompounds or mixtures of compounds on the binding of the amphipathichelix or a protein containing it with a membrane preparation can beevaluated. Techniques similar to those described above may be used—e.g.,the membrane preparation may be labeled and the amphipathic helix orprotein containing it coupled to solid support or displayed using phage.The coupling of the label to solid support containing the helix orrelevant protein or to the phage displayed helix in the presence andabsence of compound or mixtures of compounds can be determined. Theseassays can be conducted in homogeneous format by using fluorescencelabeling, for example, of the amphipathic helix. A wide variety of suchprotocols is known in the art, and the essential feature isdetermination of the effect of the compound or a mixture of compounds onthe binding of the amphipathic helix or a protein containing it and themembrane preparation. Perhaps a particularly convenient embodiment ofthis method would involve a fusion between the amphipathic helix andgreen fluorescent protein which could be produced very convenientlyusing recombinant techniques; assessment of the binding of this fusionto the membrane preparation either in a heterogeneous or homogeneousformat can then be performed.

As further described below, in Example 3, an assay for binding of theamphipathic helix to the microsomal membrane can be performed bytreating a microsomal or cytoplasmic membrane preparation in vitro witha peptide containing the helix and distributing the contents of thereaction mixture in a sedimentation gradient. The helix coupled tomembrane will reside in a relatively low density portion of the gradientand is thus separated from mere debris that is found at the bottom ofthe gradient. The floated helix can be detected in the appropriategradient fraction using polyacrylamide gel electrophoresis. The helixmay be labeled for example, with a radioisotope or a coupled fluorescentlabel such as green fluorescent protein used in a fusion. This assay canbe used to screen for compounds or protocols that disrupt binding to themicrosomal membrane by conducting the assay in the presence and absenceof the protocols or compounds and comparing the results.

In addition to the above-described methods that can be conductedextracellularly, the effect of various protocols and compounds ormixtures of compounds on the behavior in terms of binding to cytoplasmicmembranes of the relevant amphipathic helix can be determined in avariety of intracellular assays. Any eukaryotic cells may be used, buttypically and most conveniently, mammalian cells or yeast cells are usedin these assays.

Intracellular assays can be performed by generating desired peptideconstructs intracellularly from recombinant expression systems.Alternatively, the assays can be conducted by first preparing thelabeled amphipathic helix or protein containing said helix andintroducing the derivatized helix into the cells using cell-penetratingpeptides. The compounds to be tested may be introduced in the samemanner. Such cell-penetrating peptides are described, for example, in areview article by Lindgrin, M., et al., in TiPS (2000) 21:99-103, thecontents of which are incorporated herein by reference to describe anexemplary list of such cell-penetrating peptides. These peptides can becoupled to any substance to facilitate the entry of said substance intoa eukaryotic cell.

In another embodiment, MTS is able to transport both peptides andfull-length proteins into the cytoplasm of cells with or withoutcovalent linkage or crosslinking. (For example, Reference 14.) It isbelieved that the separate peptides form a noncovalent complex insolution that is then transported into the cell. In one embodiment, thepeptides are NS4B or NS5A.

The ability to deliver peptides and proteins into the intracellularmilieu for investigational studies or therapeutic applications haspreviously been limited by the lack of consistent and efficient uptakeacross the cell membrane. However, a number of reports in the lastcouple of years have identified various membrane translocation sequences(For example, see references 10, 13, 14, 16, 17, 20, 21, 22) that allowthe transduction of peptides across the plasma membrane. The mechanismof transduction is not yet clear, but it has been shown that it is notdue to receptor-mediated uptake, or in conjunction with a knowntransport mechanism. In the initial reports, the MTS was covalentlylinked to the peptide or protein that was to be delivered into the cell,but recently an MTS was identified that was able to transport bothpeptides and full-length proteins into the cytoplasm of cells in theabsence of covalent linkage or crosslinking (14). Apparently, theseparate peptides form a noncovalent complex in solution that is thentransported into the cell.

In the most direct forms of such assays, the change in intracellulardistribution of the labeled amphipathic helix either supplied per se orin the context of a larger protein can be determined. A wide variety oflabels can be used; perhaps the most convenient is a fusion with greenfluorescent protein, or simple coupling of the amphipathic helix or theprotein containing it to a detectable fluorescent label. The location ofthe labeled helix or protein containing it can then be observed by avariety of methods including direct observation and histologicaltechniques. Thus, in one example, the effect of a candidate compound orprotocol directly on the ability of the helix to bind to cellularmembranes can be assessed by coupling the helix to a reporter which isdetectable; most convenient are labels which are fusion proteins formedwith the helix, such as green fluorescent protein. The intracellularlocations of the reporter in the presence and absence of the candidateprotocol compound can then be compared. The corresponding labeled helix,which has been mutated to disrupt the amphipathic helical conformation,can conveniently be used as a control. Compounds or protocols that areable to disrupt the binding of the helix with the membrane can readilybe identified when their presence results in less label associated withcell membranes.

If histological techniques are used, the label can be less direct; forexample, the helix might be fused to a protein that can be detected witha labeled antibody or may be fused to an enzyme that can be detected inthe presence of a substrate. Once the cells are fixed histologically,the supplementary reagents can be added for detection.

Another embodiment of this uses cells harboring a vector or plasmidexpression NS5A or HCV replicon with a wild-type amphipathic helix inNS5A. The cells are exposed to a control or candidate inhibitor. At theend of the assay, the cells are fixed and stained for NS5A by indirectimmunofluorescence. A compound is deemed inhibitory if it alters thenormal membrane associated staining of NS5A.

Test compounds that appear to interrupt the ability of the amphipathichelix to bind to the cellular membranes, or which are shown to disruptthe normal binding pattern of the amphipathic helix can be confirmed asanti-HCV agents in a standard colony formation assay. In this assay, anHCV replicon is supplied with a drug resistance marker, such as neomycinresistance. When neomycin-sensitive cells are infected with the repliconand grown on neomycin-containing medium or G418, for example, only cellswherein replication can occur will be able to form colonies. Thediagnostic replicon can be used to infect neomycin-sensitive cells inthe presence and absence of the compound that has been shown to disruptthe amphipathic helix/membrane binding, and such compounds result indestroying or decreasing the ability of the infected cells to formcolonies. Alternatively, RNA replicon levels can be measured to identifythe effect of the compound.

When intracellular formats are used, in addition to testing compoundsand mixtures of compounds, as noted above, it is possible to test theeffect of protocols which involve various regimes of treatment,including, in addition to providing compounds, various antisensetechniques, and various forms of environmental stress such as pHchanges, temperature changes, mechanical disturbances and the like.

In addition to the foregoing, somewhat more sophisticated methods toassess the impact of compounds and protocols on the intracellularlocation of substances containing the amphipathic helices derived fromHepatitis C nonstructural proteins can be employed. For example, it isdemonstrated below that disruption of the amphipathic helix in NS5A, inaddition to causing the protein to fail to associate with cytoplasmicmembranes, directs this protein to the nucleus. Therefore, the assaymethods can include features in the substance containing the amphipathichelix that act as nuclear localization signals (NLS), many of which arewell known in the art. Under these conditions the label can constitute afunctionality which exerts its effect in the nucleus, and the disruptionof binding of the amphipathic helix with the cytoplasmic membrane can bedetected by a reporter function associated with the helix which exertsits effects in the nucleus. Such effects would be, for example,enhancement or repression of the expression of a detectable protein;this would involve including transactivators, transcription factors, orrepressors in the construct containing the helix and the NLS. Of course,the nuclear localization signal-derived constructs can also be detectedin the nucleus directly, using a direct detectable label of the sortdescribed above—e.g., GFP, a partner in an antibody/antigen interaction,or an enzymatic activity, or a radiolabel.

For example, in one preferred format, the amphipathic helix or a largerprotein comprising said helix is coupled to a nuclear localizationsignal and to a transactivator. The host cell is modified to contain anexpression system for a detectable protein, for example, greenfluorescent protein in its genome. The transactivator then can effectthe expression of the green fluorescent protein under conditions whereinthe protocol or compound to be tested disrupts the binding of theamphipathic helix with the cytoplasmic membrane, thus permitting thenuclear localization signal to transport the construct to the nucleuswhere the transactivator can effect expression of the GFP. Alternativeproteins whose expression can be detected are those, such as GFP, thatare detectable per se or may be detectable by virtue of their effects oncellular growth, such as his3, leu2, β-galactosidase, β-glucoronidase,SV40T antigen, chloramphenicol acetyl transferase (CAT), hygromycin Bphosphotransferase, SEAP or cell surface antigens such as CD4. Inaddition to transactivators, transcription factors which enhanceexpression include derivatives of lexA, cI, or gal4 DNA binding domainsfused to activation domains of B42, VP16 and the like.

Again, the essential feature is the detection of the effect of theproposed protocol, compounds or mixtures of compounds on the interactionof the amphipathic helix, whether supplied per se or in the context of alarger protein, with the cytoplasmic membranes. The foregoing suggestedassay formats are merely exemplary and a wide variety of such formatswill be apparent to one of skill in the art. Any such assay method isappropriate; the design of such assays and forms of label draws on awide literature available to the practitioner.

By conducting the foregoing assays, compounds and/or protocols areidentified which will be effective in treating HCV infection. By“treating” is meant both therapeutic and prophylactic treatment, andrefers to a desirable effect on viral load, or symptomology, or anydesirable outcome which mitigates the negative consequences of thetypical progress of HCV infection. The term “treat” is not to beconstrued to imply absolute cure or absolute prevention. Any helpfulamelioration or repression of the infection is sufficient to meet thisdefinition.

Compounds thus identified can be administered in conventional ways usingstandard pharmaceutical formulations such as those set forth inRemington's Pharmaceutical Sciences latest edition, Mack Publishing Co.,Easton, Pa., incorporated herein by reference. (See also, References23-33.) Various enteral or parenteral routes of administration may beused, including administering by injection, such as intravenous,intramuscular, subcutaneous and the like, or by oral administration orby suppository. In addition, sustained release compositions can also beused.

The terms “an effective amount,” “an anti-HCV effective amount,” or a“pharmaceutically effective amount” of a peptide of the presentinvention as applied to such molecules refers to an amount of a peptide,or combination of two or more peptides as disclosed herein, effective inreducing or ameliorating conditions, symptoms, or disorders associatedwith HCV infection or associated pathogenesis in patients, and/or inreducing viral levels in vitro or in vivo.

Effective amounts of peptides for the treatment or prevention of HCVinfections, delivery vehicles containing peptides or constructs encodingthe same, agonists, and treatment protocols, can be determined byconventional means. For example, the medical practitioner can commencetreatment with a low dose of one or more peptides in a subject orpatient in need thereof, and then increase the dosage, or systematicallyvary the dosage regimen, monitor the effects thereof on the patient orsubject, and adjust the dosage or treatment regimen to maximize thedesired therapeutic effect. Further discussion of optimization of dosageand treatment regimens can be found in Benet et al., in Goodman &Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition,Hardman et al., Eds., McGraw-Hill, New York, (1996), Chapter 1, pp.3-27, and L. A. Bauer, in Pharmacotherapy, A Pathophysiologic Approach,Fourth Edition, DiPiro et al., Eds., Appleton & Lange, Stamford, Conn.,(1999), Chapter 3, pp. 21-43, and the references cited therein, to whichthe reader is referred.

The dosage levels and mode of administration will be dependent on thenature of the compound identified and the particular situation of thesubject. Optimization of routes of administration, dosage levels, andadjustment of protocols, including monitoring systems to assesseffectiveness of the treatment are routine matters well within ordinaryskill. Protocols which are identified using the intracellular assays setforth above must, of course, be modified in the context of treatment ofsubjects, and this, too, falls within the skill of the practitioner.

Typically, the compounds that are identified by the methods of theinvention will be “small molecules”—i.e., synthetic organic structurestypical of pharmaceuticals. Examples of such “small molecules” arefound, for example, in the Physicians' Desk Reference (with respect toapproved drugs), the Merck Index and the U.S. Pharmacopoeia. However,such compounds may also include peptides, peptidomimetics, nucleicacids, peptide nucleic acids, carbohydrates, lipids, and the like. Asnoted above, the HCV nonstructural protein amphipathic helix used inthese assays may be the native helix, both alone and in the context of alarger protein and the assays may also utilize a homologous form of thehelix included within the definition above which retains the spatial andcharge configuration of the native form.

In addition to compounds identified by means of the foregoing assays,protocols and substances useful in treating HCV infection can beformulated and utilized a priori. For example, the helix itself, or afunctional fragment thereof can readily be used in treatment by virtueof its ability to bind the helix as it resides in an HCV non-structuralprotein itself or to compete with the helix as it resides in an HCVnon-structural protein itself for sites on the cytoplasmic membrane towhich the helix will bind. Thus, formulations of the HCV nonstructuralprotein amphipathic helix or functional fragment thereof can be useddirectly to treat HCV infection. Typically, these formulations willcontain peptides bearing the helix of less than 60 amino acids, inanother embodiment less than 50 amino acids, in another embodiment lessthan 30 amino acids, and, in yet another embodiment less than 25 aminoacids in another embodiment, not be less than 4 amino acids, and may, inaddition to the peptide region bearing the helix, further containcomponents to enhance membrane penetration. The peptides comprising thehelix may be as short as 10-15 amino acids, 6-9 amino acids or 4-6 aminoacids, so long as functionality in terms of competitive binding, orinhibition of binding, is retained. Methods to synthesize such peptidesare, of course, well known, both direct and recombinant methods may beused. Thus, a peptide useful in the method of the invention as to itshelix-based antiviral activity will typically contain from about 4 to 60amino acids, and certainly far less than the several hundred amino acidscontained in the native viral protein from which it can be derived. Thisrelatively short peptide may optionally be coupled to additionalsequence either for labeling, or, more typically, for conferring theability to enter cells. Such membrane penetration facilitators areknown, for example, the HIV-tat peptide or MTSs described herein, areable to introduce non-native proteins into cells. The peptides to beadministered will thus contain a portion comprising the amphipathichelix and a heterologous portion for facilitating cellular entry. Entrycan also be facilitated by liposomes or cationic polymers, for example.However, wild type NS5A is able to cross the membrane without theassistance of a facilitator. Wild type NS5A is capable of entering thecell without any previously described MTS peptide or other facilitator.

As used herein “functional” refers to a protein, peptide, helix orantibody, or fragment thereof, which possesses the ability to inhibitthe binding of a nonstructural HCV protein, for example, NS4B or NS5A,to a cell membrane.

The peptides useful in the methods of the invention can also begenerated in the subject to be treated by virtue of administeringexpression systems for those peptides consisting entirely ofgene-encoded amino acids. These expression systems may be introduced asnaked DNA, as expression vectors suitable for transfection of mammaliancells, or preferably using adenoviral or retroviral or other suitableviral vectors.

As described above, however, these competitor peptides need not be thenative sequences per se and need not even be peptides per se, but maycontain isosteric linkages or other polymeric features that result insimilar charge/shape features as compared to the native helices.

Peptides, or compounds with similar charge/shape features and having theactivity of the peptides described herein, can be identified by phagedisplay using wild-type amphipathic helix and a mutant amphipathic helixpeptides as positive and negative selectors, respectively.

The compositions or agents of the invention may comprise, consistessentially of, or consist of the peptide sequences disclosed herein.The phrase “consists essentially of or consisting essentially of” or thelike, when applied to anti-HCV peptides encompassed by the presentinvention refers to peptide sequences like those disclosed herein, butwhich contain additional amino acids (or analogs or derivatives thereofas discussed above). Such additional amino acids, etc., however, do notmaterially affect the basic and novel characteristic(s) of thesepeptides in modulating, attenuating, or inhibiting HCV infection,replication, and/or pathogenesis, including the specific quantitativeeffects of these peptides, compared to those of the correspondingpeptides disclosed herein.

In one approach, the agent may be a transdominant inhibitor of themembrane association function whereby forms of the amphipathic helixthat interfere with the ability of the helix to form oligomers can beused. Thus, by generating or providing a mutant form of the helixcontaining one or more amino acid substations, this form may associatewith the native helix to provide an inactive form or rendering it unableto dimerize or oligomerize with additional native forms. In oneapproach, the decoy peptide is mutated to convert hydrophobic aminoacids to hydrophilic ones thus destroying the hydrophobic face of thehelix. For example, mutated versions of the peptide sequence for NS5Astrains would include

-   SGSWLRDDWDWECEVLSDDKTWLKAK (SEQ ID NO: 15) or-   SGSWLRDDWDWECTVLTDDKTWLQSKL (SEQ ID NO: 16). SEQ ID NOS: 15 and 16    are used as PEP2 in Example 4, below.

In another approach, the agent is a competitive inhibitor of theamphipathic helix. These competitive inhibitors may interrupt thebinding between the helix and the membrane, achieving the desiredeffect. Such inhibitors may be fragments of the wild type sequence ofthe amphipathic sequence or variants or mutants thereof. Fragments ofthe HCV nonstructural proteins that may be used as competitiveinhibitors may include, but are not limited to:

(NS4B 6-34) (SEQ ID NO: 462) YIEQGMMLAEQFKQKALGLLQTASRHAEV, (NS5B 65-87)(SEQ ID NO: 3) QDVLKEVKAAASKVKANLLSVEE, and (NS5B 107-125)(SEQ ID NO: 4) DVRCHARKAVAHINSVWKD.

Another competitive inhibitor of NS5A would include:SGSWLRDVWDWICTVLTDFKTWLQSKL (SEQ ID NO: 14) and variants and mutantsthereof. Variants and mutants of this peptide would include a peptidewith one or more of the following amino acid substitutions: substitutionof L at amino acid 16 by A or K; or substitution of the T at amino acid17 by A; or substitution of the D at amino acid 18 by A; or substitutionof the F at amino acid 19 by A; or substitution of the K at amino acid20 by A; or substitution of the W at amino acid 22 by A; or substitutionof the L at amino acid 23 by K, and derivatives thereof. Another suchmutant of NSSA is: SGSX₁X₂X₃X₅X₆ X₇ X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀QSK L (SEQ ID NO:467), where X₁ is W,A or F; X2 is L, A or K; X3 is R, A or N; X4 is D, A or S; X₅ is V, I, Dor S; X₆ is W, A or F; X₇ is D, A or S; X₈ is W, A or F; X₉ is I, E orL; X₁₀ is C, A or S; X₁₁is T, E, A or W; X₁₂ is V, D or S; X₁₃ is L, Aor K; X₁₄ is T, S, A or W; X₁₅ is D, A or S; X₁₆ is F, D, or L; X₁₇ isK, A or L; X₁₈ is T, A or W; X₁₉ is W, A or F; and X₂₀ is L, A or K. Inanother embodiment, X₁, X₂, X₄, X₆, X₈, X₁₀, X₁₃, X₁₅, X₁₆ or X₂₀ may beD or X₁₆ is A. Where the agent is a competitive inhibitor, some of theabove mutations would enhance the inhibitory activity of the peptide,others would completely or partially abolish the inhibitory activity ofthe peptide. Mutations may be observed alone or in combination, forexample, any one substitution, X₁₋₂₀, occurs within the context of thewild type sequence at any one time, a combination of two mutations suchas: X₅ and X₉, X₅ and X₁₆, or X₉ and X₁₆ or three mutations are found inthe same peptide, X₅, with X₉, and X₁₆. Additional mutations of theabove inhibitor may include the sequence:

-   X₁X₂DVWDWICTX₃X₄X₅X₆X₇X₈X₉(SEQ ID NO:468) wherein when X₁ and X₂ are    present, X₁ is L and X₂ is R; wherein X₁ and X₂ are present, X₃, X₄,    X₅, X₆, X₇, X₈ and X₉ are optionally present, and when X₃, X₄, X₅,    X₆, X₇, X₈ and X₉ are present, X₃ is V, X₄ is L, X₅ is T, X₆ is D,    X₇ is F, X₈ is K and X₉ is T; and wherein X₆ is present, X3, X₄ and    X₅ are all present, wherein X₆ is D, X₃ is V, X₄ is L, and X₅ is T.    In particular where the sequence is LRDVWDWICTVLTDFKT (SEQ ID NO:    463), LRDVWDWICT (SEQID NO: 464) or DVWDWICTVLTD (SEQ ID NO: 465).    In another embodiment, the competitive inhibitor may be SWLRDVWDWIC    (SEQ ID NO: 466).

A mutant, and competitive inhibitor, of NS4B may include the sequence:X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉X₂₀X₂₁X₂₂X₂₃X₂₄X₂₅X₂₆X₂₇X₂₈X₂₉where X₁ is Y, A or H; X₂ is I, E or L; X₃ is E, A or Q; X₄ is Q, V orR; X₅ is G, A or D; X₆ is M, E or C; X₇ is M, E or C; X₈ is L, E or Q;X₉ is A, D or S; X₁₀ is E, A or Q; X₁₁ is Q, V or R; X₁₂ is F, D or L;X₁₃ is K, I or Q; X₁₄ is Q, V or R; X₁₅ is K, I or Q; X₁₆ is A, D or G;X₁₇ is L, A or K; X₁₈ is G, E or S; X₁₉ is L, A or K; X₂₀ is L, A or K;X₂₁ is Q, V or R; X₂₂ is T, A or W; X₂₃ is A, D or G; X₂₄ is S, D or A;X₂₅ is R, L or W; X₂₆ is Q, V or R; X₂₇ is A, D or G; and X₂₈ is E, A orQ.

While the majority of the above mutations and substitutions areconservative (i.e. wild type hydrophobic residues are substituted withadditional hydrophobic residues (for example, A to F), charged residuesare substituted with similarly charged residues, etc.), it is noted thatnonconservative substitutions may also be performed. For example, wherea wild type hydrophobic residue is substituted with a hydrophilicresidue, or a negatively charged residue (for example, D) is substitutedwith a positively charged residue (for example, K).

Where the agent is a competitive inhibitor, some of the above mutationswould enhance the inhibitory activity of the peptide, others wouldcompletely or partially abolish the inhibitory activity of the peptide.Mutations may be observed alone or in combination, for example, any onesubstitution, X₁₋₂₈, occurs within the context of the wild type sequenceat any one time, a combination of two mutations such as: X₂ and X₅, X₂and X₁₉, or X₅ and X₁₉ or three mutations are found in the same peptide,X₂, with X₅, and X₁₉.

In another embodiment, the agent may be a complementary peptide to thehelix. Complementary peptides may interrupt the binding between thehelix and the membrane, achieving the desired effect. Such complementarypeptides may also inhibit the formation of the amphipathic helix, mayinteract or bind to the helix to inhibit binding of the helix tocellular membranes, or may otherwise inhibit the amphipathic helices.

For example, the HCV genomic RNA sequence that codes for the NS4Bamphipathic helix in the HCV genotype 1A sequence is:

(SEQ ID NO: 17) UACAUCGAGCAAGGGAUGAUGCUCGCTGAGCAGUUCAAGCAGAAGGCCCUCGGCCUCCUGCAGACCGCGUCCCGCCAAGCAGAG.(Kolykhalov, A. A. and Rice, C. M., Science 277 (5325), 570-574 (1997);GenBank Accession No.: AF009606.) The protein translation of the abovesequence is:YIEQGMMLAEQFKQKALGLLQTASRQAE (SEQ ID NO: 18). The reverse complementcDNA sequence corresponding to the HCV genomic RNA sequence that codesfor the NS4B amphipathic helix (GenBank Accession No.: AF009606) is:CTCTGCTTGGCGGGACGCGGTCTGCAGGAGGCCGAGGGCCTTCTGCTTGAACTGCTCAGCGAGCATCATCCCTTGCTCGATGTA (SEQ ID NO: 19). The complementarypeptide translated from the reverse complement sequence is:LCLAGRGLQEAEGLLLELLSEHHPLLDV (SEQ ID NO: 20). This complementarypeptide, as a whole, or in part, may be active as an HCV antiviral ormay be useful in the prediction of small molecules that are HCVantivirals. In one embodiment a fragment of this complementary peptidecomprising 6-27 amino acids may be used in the discovery of HCVantivirals. Table 1 sets forth exemplary peptides (SEQ ID NOS: 21-295)of this embodiment.

TABLE 1 All smaller NS4B complementary peptides with6 or more amino acids: Sequence Identification Sequence Number LQEAEGSEQ ID NO: 21 CLAGRG SEQ ID NO: 22 LELLSE SEQ ID NO: 23 GLQEAESEQ ID NO: 24 LLELLS SEQ ID NO: 25 QEAEGL SEQ ID NO: 26 RGLQEASEQ ID NO: 27 LAGRGL SEQ ID NO: 28 GRGLQE SEQ ID NO: 29 ELLSEHSEQ ID NO: 30 LLLELL SEQ ID NO: 31 LCLAGR SEQ ID NO: 32 HPLLDVSEQ ID NO: 33 AEGLLL SEQ ID NO: 34 AGRGLQ SEQ ID NO: 35 EAEGLLSEQ ID NO: 36 LSEHHP SEQ ID NO: 37 HHPLLD SEQ ID NO: 38 EGLLLESEQ ID NO: 39 SEHHPL SEQ ID NO: 40 LLSEHH SEQ ID NO: 41 EHHPLLSEQ ID NO: 42 GLLLEL SEQ ID NO: 43 LELLSEH SEQ ID NO: 44 ELLSEHHSEQ ID NO: 45 LLELLSE SEQ ID NO: 46 LLSEHHP SEQ ID NO: 47 HHPLLDVSEQ ID NO: 48 GLLLELL SEQ ID NO: 49 LAGRGLQ SEQ ID NO: 50 LSEHHPLSEQ ID NO: 51 LCLAGRG SEQ ID NO: 52 LQEAEGL SEQ ID NO: 53 AGRGLQESEQ ID NO: 54 AEGLLLE SEQ ID NO: 55 GRGLQEA SEQ ID NO: 56 RGLQEAESEQ ID NO: 57 QEAEGLL SEQ ID NO: 58 SEHHPLL SEQ ID NO: 59 EHHPLLDSEQ ID NO: 60 GLQEAEG SEQ ID NO: 61 EGLLLEL SEQ ID NO: 62 LLLELLSSEQ ID NO: 63 EAEGLLL SEQ ID NO: 64 CLAGRGL SEQ ID NO: 65 CLAGRGLQSEQ ID NO: 66 QEAEGLLL SEQ ID NO: 67 LAGRGLQE SEQ ID NO: 68 LCLAGRGLSEQ ID NO: 69 AGRGLQEA SEQ ID NO: 70 LLLELLSE SEQ ID NO: 71 LELLSEHHSEQ ID NO: 72 AEGLLLEL SEQ ID NO: 73 EGLLLELL SEQ ID NO: 74 LLSEHHPLSEQ ID NO: 75 GRGLQEAE SEQ ID NO: 76 EAEGLLLE SEQ ID NO: 77 RGLQEAEGSEQ ID NO: 78 LLELLSEH SEQ ID NO: 79 SEHHPLLD SEQ ID NO: 80 LSEHHPLLSEQ ID NO: 81 LQEAEGLL SEQ ID NO: 82 ELLSEHHP SEQ ID NO: 83 GLLLELLSSEQ ID NO: 84 EHHPLLDV SEQ ID NO: 85 GLQEAEGL SEQ ID NO: 86 LLSEHHPLLSEQ ID NO: 87 LELLSEHHP SEQ ID NO: 88 RGLQEAEGL SEQ ID NO: 89 ELLSEHHPLSEQ ID NO: 90 LCLAGRGLQ SEQ ID NO: 91 AGRGLQEAE SEQ ID NO: 92 GLLLELLSESEQ ID NO: 93 LSEHHPLLD SEQ ID NO: 94 EGLLLELLS SEQ ID NO: 95 GLQEAEGLLSEQ ID NO: 96 AEGLLLELL SEQ ID NO: 97 QEAEGLLLE SEQ ID NO: 98 SEHHPLLDVSEQ ID NO: 99 LLLELLSEH SEQ ID NO: 100 GRGLQEAEG SEQ ID NO: 101CLAGRGLQE SEQ ID NO: 102 EAEGLLLEL SEQ ID NO: 103 LAGRGLQEASEQ ID NO: 104 LLELLSEHH SEQ ID NO: 105 LQEAEGLLL SEQ ID NO: 106LSEHHPLLDV SEQ ID NO: 107 LELLSEHHPL SEQ ID NO: 108 RGLQEAEGLLSEQ ID NO: 109 LLSEHHPLLD SEQ ID NO: 110 AEGLLLELLS SEQ ID NO: 111GLQEAEGLLL SEQ ID NO: 112 EGLLLELLSE SEQ ID NO: 113 LCLAGRGLQESEQ ID NO: 114 LLLELLSEHH SEQ ID NO: 115 EAEGLLLELL SEQ ID NO: 116GLLLELLSEH SEQ ID NO: 117 ELLSEHHPLL SEQ ID NO: 118 GRGLQEAEGLSEQ ID NO: 119 LQEAEGLLLE SEQ ID NO: 120 LLELLSEHHP SEQ ID NO: 121CLAGRGLQEA SEQ ID NO: 122 QEAEGLLLEL SEQ ID NO: 123 AGRGLQEAEGSEQ ID NO: 124 LAGRGLQEAE SEQ ID NO: 125 LLELLSEHHPL SEQ ID NO: 126CLAGRGLQEAE SEQ ID NO: 127 RGLQEAEGLLL SEQ ID NO: 128 LAGRGLQEAEGSEQ ID NO: 129 LQEAEGLLLEL SEQ ID NO: 130 LLLELLSEHHP SEQ ID NO: 131ELLSEHHPLLD SEQ ID NO: 132 AGRGLQEAEGL SEQ ID NO: 133 LLSEHHPLLDVSEQ ID NO: 134 LGLAGRGLQEA SEQ ID NO: 135 GLLLELLSEHH SEQ ID NO: 136LELLSEHHPLL SEQ ID NO: 137 QEAEGLLLELL SEQ ID NO: 138 EGLLLELLSEHSEQ ID NO: 139 GRGLQEAEGLL SEQ ID NO: 140 AEGLLLELLSE SEQ ID NO: 141GLQEAEGLLLE SEQ ID NO: 142 EAEGLLLELLS SEQ ID NO: 143 EGLLLELLSEHHSEQ ID NO: 144 RGLQEAEGLLLE SEQ ID NO: 145 GLLLELLSEHHP SEQ ID NO: 146LQEAEGLLLELL SEQ ID NO: 147 GLQEAEGLLLEL SEQ ID NO: 148 LAGRGLQEAEGLSEQ ID NO: 149 GRGLQEAEGLLL SEQ ID NO: 150 QEAEGLLLELLS SEQ ID NO: 151AEGLLLELLSEH SEQ ID NO: 152 AGRGLQEAEGLL SEQ ID NO: 153 LELLSEHHPLLDSEQ ID NO: 154 LCLAGRGLQEAE SEQ ID NO: 155 LLLELLSEHHPL SEQ ID NO: 156LLELLSEHHPLL SEQ ID NO: 157 ELLSEHHPLLDV SEQ ID NO: 158 EAEGLLLELLSESEQ ID NO: 159 CLAGRGLQEAEG SEQ ID NO: 160 CLAGRGLQEAEGL SEQ ID NO: 161RGLQEAEGLLLEL SEQ ID NO: 162 LLELLSEHHPLLD SEQ ID NO: 163 GRGLQEAEGLLLESEQ ID NO: 164 LCLAGRGLQEAEG SEQ ID NO: 165 LELLSEHHPLLDV SEQ ID NO: 166EGLLLELLSEHHP SEQ ID NO: 167 EAEGLLLELLSEH SEQ ID NO: 168 GLQEAEGLLLELLSEQ ID NO: 169 AGRGLQEAEGLLL SEQ ID NO: 170 AEGLLLELLSEHH SEQ ID NO: 171LAGRGLQEAEGLL SEQ ID NO: 172 QEABGLLLELLSE SEQ ID NO: 173 LLLELLSEHHPLLSEQ ID NO: 174 LQEAEGLLLELLS SEQ ID NO: 175 GLLLELLSEHHPL SEQ ID NO: 176EAEGLLLELLSEHH SEQ ID NO: 177 EGLLLELLSEHHPL SEQ ID NO: 178AGRGLQEAEGLLLE SEQ ID NO: 179 LCLAGRGLQEAEGL SEQ ID NO: 180GLLLELLSEHHPLL SEQ ID NO: 181 QEAEGLLLELLSEH SEQ ID NO: 182RGLQEAEGLLLELL SEQ ID NO: 183 LLLELLSEHHPLLD SEQ ID NO: 184LQEAEGLLLELLSE SEQ ID NO: 185 LLELLSEHHPLLDV SEQ ID NO: 186GLQEAEGLLLELLS SEQ ID NO: 187 GRGLQEAEGLLLEL SEQ ID NO: 188AEGLLLELLSEHHP SEQ ID NO: 189 CLAGRGLQEAEGLL SEQ ID NO: 190LAGRGLQEAEGLLL SEQ ID NO: 191 LLLELLSEHHPLLDV SEQ ID NO: 192AGRGLQEAEGLLLEL SEQ ID NO: 193 AEGLLLELLSEHHPL SEQ ID NO: 194GLLLELLSEHHPLLD SEQ ID NO: 195 LQEAEGLLLELLSEH SEQ ID NO: 196GLQEAEGLLLELLSE SEQ ID NO: 197 LCLAGRGLQEAEGLL SEQ ID NO: 198EAEGLLLELLSEHHP SEQ ID NO: 199 GRGLQEAEGLLLELL SEQ ID NO: 200QEAEGLLLELLSEHH SEQ ID NO: 201 LAGRGLQEAEGLLLE SEQ ID NO: 202GLAGRGLQEAEGLLL SEQ ID NO: 203 EGLLLELLSEHHPLL SEQ ID NO: 204RGLQEAEGLLLELLS SEQ ID NO: 205 LAGRGLQEAEGLLLEL SEQ ID NO: 206GLLLELLSEHHPLLDV SEQ ID NO: 207 GRGLQEAEGLLLELLS SEQ ID NO: 208AGRGLQEAEGLLLELL SEQ ID NO: 209 GLQEAEGLLLELLSEH SEQ ID NO: 210RGLQEAEGLLLELLSE SEQ ID NO: 211 LQEAEGLLLELLSEHH SEQ ID NO: 212CLAGRGLQEAEGLLLE SEQ ID NO: 213 QEAEGLLLELLSEHHP SEQ ID NO: 214AEGLLLELLSEHHPLL SEQ ID NO: 215 EAEGLLLELLSEHHPL SEQ ID NO: 216LCLAGRGLQEAEGLLL SEQ ID NO: 217 EGLLLELLSEHHPLLD SEQ ID NO: 218LCLAGRGLQEAEGLLLE SEQ ID NO: 219 QEAEGLLLELLSEHHPL SEQ ID NO: 220EAEGLLLELLSEHHPLL SEQ ID NO: 221 RGLQEAEGLLLELLSEH SEQ ID NO: 222CLAGRGLQEAEGLLLEL SEQ ID NO: 223 GRGLQEAEGLLLELLSE SEQ ID NO: 224LAGRGLQEAEGLLLELL SEQ ID NO: 225 AGRGLQEAEGLLLELLS SEQ ID NO: 226GLQEAEGLLLELLSEHH SEQ ID NO: 227 EGLLLELLSEHHPLLDV SEQ ID NO: 228AEGLLLELLSEHHPLLD SEQ ID NO: 229 LQEAEGLLLELLSEHHP SEQ ID NO: 230CLAGRGLQEAEGLLLELL SEQ ID NO: 231 LQEAEGLLLELLSEHHPL SEQ ID NO: 232GRGLQEAEGLLLELLSEH SEQ ID NO: 233 EAEGLLLELLSEHHPLLD SEQ ID NO: 234AEGLLLELLSEHHPLLDV SEQ ID NO: 235 GLQEAEGLLLELLSEHHP SEQ ID NO: 236RGLQEAEGLLLELLSEHH SEQ ID NO: 237 QEAEGLLLELLSEHHPLL SEQ ID NO: 238LAGRGLQEAEGLLLELLS SEQ ID NO: 239 LCLAGRGLQEAEGLLLEL SEQ ID NO: 240AGRGLQEAEGLLLELLSE SEQ ID NO: 241 AGRGLQEAEGLLLELLSEH SEQ ID NO: 242GLQEAEGLLLELLSEHHPL SEQ ID NO: 243 GRGLQEAEGLLLELLSEHH SEQ ID NO: 244LGLAGRGLQEAEGLLLELL SEQ ID NO: 245 CLAGRGLQEAEGLLLELLS SEQ ID NO: 246RGLQEAEGLLLELLSEHHP SEQ ID NO: 247 LAGRGLQEAEGLLLELLSE SEQ ID NO: 248QEAEGLLLELLSEHHPLLD SEQ ID NO: 249 LQEAEGLLLELLSEHHPLL SEQ ID NO: 250EAEGLLLELLSEHHPLLDV SEQ ID NO: 251 RGLQEAEGLLLELLSEHHPL SEQ ID NO: 252GLQEAEGLLLELLSEHHPLL SEQ ID NO: 253 AGRGLQEAEGLLLELLSEHH SEQ ID NO: 254LAGRGLQEAEGLLLELLSEH SEQ ID NO: 255 GRGLQEAEGLLLELLSEHHP SEQ ID NO: 256LCLAGRGLQEAEGLLLELLS SEQ ID NO: 257 LQEABGLLLELLSEHHPLLD SEQ ID NO: 258QEAEGLLLELLSEHHPLLDV SEQ ID NO: 259 CLAGRGLQEAEGLLLELLSE SEQ ID NO: 260LCLAGRGLQEAEGLLLELLSE SEQ ID NO: 261 RGLQEAEGLLLELLSEHHPLLSEQ ID NO: 262 LQEAEGLLLELLSEHHPLLDV SEQ ID NO: 263GRGLQEAEGLLLELLSEHHPL SEQ ID NO: 264 AGRGLQEAEGLLLELLSEHHPSEQ ID NO: 265 LAGRGLQEAEGLLLELLSEHH SEQ ID NO: 266GLAGRGLQEAEGLLLELLSEH SEQ ID NO: 267 GLQEAEGLLLELLSEHHPLLDSEQ ID NO: 268 GLQEAEGLLLELLSEHHPLLDV SEQ ID NO: 269AGRGLQEAEGLLLELLSEHHPL SEQ ID NO: 270 CLAGRGLQEAEGLLLELLSEHHSEQ ID NO: 271 GRGLQEAEGLLLELLSEHHPLL SEQ ID NO: 272RGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 273 LAGRGLQEAEGLLLELLSEHHPSEQ ID NO: 274 LCLAGRGLQEAEGLLLELLSEH SEQ ID NO: 275CLAGRGLQEAEGLLLELLSEHHP SEQ ID NO: 276 GRGLQEAEGLLLELLSEHHPLLDSEQ ID NO: 277 LCLAGRGLQEAEGLLLELLSEHH SEQ ID NO: 278LAGRGLQEAEGLLLELLSEHHPL SEQ ID NO: 279 RGLQEAEGLLLELLSEHHPLLDVSEQ ID NO: 280 AGRGLQEAEGLLLELLSEHHPLL SEQ ID NO: 281AGRGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 282 CLAGRGLQEAEGLLLELLSEHHPLSEQ ID NO: 283 LCLAGRGLQEAEGLLLELLSEHHP SEQ ID NO: 284GRGLQEAEGLLLELLSEHHPLLDV SEQ ID NO: 285 LAGRGLQEAEGLLLELLSEHHPLLSEQ ID NO: 286 AGRGLQEAEGLLLELLSEHHPLLDV SEQ ID NO: 287LCLAGRGLQEAEGLLLELLSEHHPL SEQ ID NO: 288 LAGRGLQEAEGLLLELLSEHHPLLDSEQ ID NO: 289 CLAGRGLQEAEGLLLELLSEHHPLL SEQ ID NO: 290LCLAGRGLQEAEGLLLELLSEHHPLL SEQ ID NO: 291 LAGRGLQEAEGLLLELLSEHHPLLDVSEQ ID NO: 292 CLAGRGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 293LCLAGRGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 294 CLAGRGLQEAEGLLLELLSEHHPLLDVSEQ ID NO: 295

In a particular embodiment, the complementary peptides of the inventionare used, in whole or in part, in the prediction of small molecules thatare HCV antivirals. “Small molecule” as defined above, may includesynthetic organic structures typical of pharmaceuticals, and may alsoinclude peptides, nucleic acids, peptide nucleic acids, carbohydrates,lipids, and the like. Additionally, small molecules, as used herein, mayinclude chemically synthesized peptidomimetics of the 6-mer to 9-merpeptides of the invention.

Additionally, the HCV genomic RNA sequence that codes for the NS5Aamphipathic helix in the HCV genotype 1A sequence is:UGGCUAAGGGACAUCUGGGACUGGAUAUGCGAGGUGCUGAGCGACUUUAAGACCUGGCUGAAAGCCAAGCUC (SEQ ID NO: 296). (Kolykhalov, A. A. and Rice,C. M., Science 277 (5325), 570-574 (1997); GenBank Accession No.:AF009606.) The protein translation of the above sequence is:WLRDIWDWICEVLSDFKTWLKAKL (SEQ ID NO: 297). The reverse complement cDNAsequence corresponding to the HCV genomic RNA sequence that codes forthe NS5A amphipathic helix (GenBank Accession No.: AF009606) is:GAGCTTGGCTTTCAGCCAGGTCTTAAAGTCGCTCAGCACCTCGCATATCCAGTCCCAGATGTCCCTTAGCCA (SEQ ID NO: 298). This reverse complement sequencecontains a stop codon at codon 23. The complementary peptide translatedfrom the reverse complement sequence is: ELGFQPGLKVAQHLAYPVPDVP (SEQ IDNO: 299). This complementary peptide, as a whole, or in part, may beactive as an HCV antiviral or may be useful in the prediction of smallmolecules that are HCV antivirals. In one embodiment a fragment of thiscomplementary peptide comprising 6-21 amino acids may is used in thediscovery of HCV antivirals. Table 2 sets forth exemplary peptides (SEQID NOS: 300-451) of this embodiment.

TABLE 2 NS5A complementary peptides with 6 or more amino acids: SequenceIdentification Sequence Number AQHLAY SEQ ID NO: 300 LGFQPGSEQ ID NO: 301 GLKVAQ SEQ ID NO: 302 ELGFQP SEQ ID NO: 303 PVPDVPSEQ ID NO: 304 FQPGLK SEQ ID NO: 305 GFQPGL SEQ ID NO: 306 VAQHLASEQ ID NO: 307 LKVAQH SEQ ID NO: 308 HLAYPV SEQ ID NO: 309 KVAQHLSEQ ID NO: 310 QPGLKV SEQ ID NO: 311 LAYPVP SEQ ID NO: 312 YPVPDVSEQ ID NO: 313 QHLAYP SEQ ID NO: 314 PGLKVA SEQ ID NO: 315 AYPVPDSEQ ID NO: 316 QPGLKVA SEQ ID NO: 317 ELGFQPG SEQ ID NO: 318 HLAYPVPSEQ ID NO: 319 LAYPVPD SEQ ID NO: 320 AQHLAYP SEQ ID NO: 321 FQPGLKVSEQ ID NO: 322 KVAQHLA SEQ ID NO: 323 LGFQPGL SEQ ID NO: 324 AYPVPDVSEQ ID NO: 325 PGLKVAQ SEQ ID NO: 326 LKVAQHL SEQ ID NO: 327 GLKVAQHSEQ ID NO: 328 VAQHLAY SEQ ID NO: 329 GFQPGLK SEQ ID NO: 330 QHLAYPVSEQ ID NO: 331 YPVPDVP SEQ ID NO: 332 AYPVPDVP SEQ ID NO: 333 PGLKVAQHSEQ ID NO: 334 QHLAYPVP SEQ ID NO: 335 LAYPVPDV SEQ ID NO: 336 LKVAQHLASEQ ID NO: 337 GLKVAQHL SEQ ID NO: 338 GFQPGLKV SEQ ID NO: 339 AQHLAYPVSEQ ID NO: 340 VAQHLAYP SEQ ID NO: 341 KVAQHLAY SEQ ID NO: 342 ELGFQPGLSEQ ID NO: 343 LGFQPGLK SEQ ID NO: 344 FQPGLKVA SEQ ID NO: 345 HLAYPVPDSEQ ID NO: 346 QPGLKVAQ SEQ ID NO: 347 ELGFQPGLK SEQ ID NO: 348VAQHLAYPV SEQ ID NO: 349 LGFQPGLKV SEQ ID NO: 350 QPGLKVAQHSEQ ID NO: 351 KVAQHLAYP SEQ ID NO: 352 PGLKVAQHL SEQ ID NO: 353FQPGLKVAQ SEQ ID NO: 354 GLKVAQHLA SEQ ID NO: 355 LAYPVPDVPSEQ ID NO: 356 HLAYPVPDV SEQ ID NO: 357 GFQPGLKVA SEQ ID NO: 358AQHLAYPVP SEQ ID NO: 359 QHLAYPVPD SEQ ID NO: 360 LKVAQHLAYSEQ ID NO: 361 QPGLKVAQHL SEQ ID NO: 362 PGLKVAQHLA SEQ ID NO: 363HLAYPVPDVP SEQ ID NO: 364 GLKVAQHLAY SEQ ID NO: 365 VAQHLAYPVPSEQ ID NO: 366 KVAQHLAYPV SEQ ID NO: 367 GFQPGLKVAQ SEQ ID NO: 368QHLAYPVPDV SEQ ID NO: 369 AQHLAYPVPD SEQ ID NO: 370 FQPGLKVAQHSEQ ID NO: 371 ELGFQPGLKV SEQ ID NO: 372 LKVAQHLAYP SEQ ID NO: 373LGFQPGLKVA SEQ ID NO: 374 QHLAYPVPDVP SEQ ID NO: 375 LKVAQHLAYPVSEQ ID NO: 376 VAQHLAYPVPD SEQ ID NO: 377 PGLKVAQHLAY SEQ ID NO: 378LGFQPGLKVAQ SEQ ID NO: 379 KVAQHLAYPVP SEQ ID NO: 380 ELGFQPGLKVASEQ ID NO: 381 GLKVAQHLAYP SEQ ID NO: 382 AQHLAYPVPDV SEQ ID NO: 383FQPGLKVAQHL SEQ ID NO: 384 QPGLKVAQHLA SEQ ID NO: 385 GFQPGLKVAQHSEQ ID NO: 386 LGFQPGLKVAQH SEQ ID NO: 387 PGLKVAQHLAYP SEQ ID NO: 388LKVAQHLAYPVP SEQ ID NO: 389 GFQPGLKVAQHL SEQ ID NO: 390 VAQHLAYPVPDVSEQ ID NO: 391 QPGLKVAQHLAY SEQ ID NO: 392 ELGFQPGLKVAQ SEQ ID NO: 393AQHLAYPVPDVP SEQ ID NO: 394 KVAQHLAYPVPD SEQ ID NO: 395 FQPGLKVAQHLASEQ ID NO: 396 GLKVAQHLAYPV SEQ ID NO: 397 LGFQPGLKVAQHL SEQ ID NO: 398VAQHLAYPVPDVP SEQ ID NO: 399 PGLKVAQHLAYPV SEQ ID NO: 400 GFQPGLKVAQHLASEQ ID NO: 401 LKVAQHLAYPVPD SEQ ID NO: 402 QPGLKVAQHLAYP SEQ ID NO: 403ELGFQPGLKVAQH SEQ ID NO: 404 GLKVAQHLAYPVP SEQ ID NO: 405 FQPGLKVAQHLAYSEQ ID NO: 406 KVAQHLAYPVPDV SEQ ID NO: 407 ELGFQPGLKVAQHLSEQ ID NO: 408 QPGLKVAQHLAYPV SEQ ID NO: 409 GFQPGLKVAQHLAYSEQ ID NO: 410 LGFQPGLKVAQHLA SEQ ID NO: 411 FQPGLKVAQHLAYPSEQ ID NO: 412 KVAQHLAYPVPDVP SEQ ID NO: 413 PGLKVAQHLAYPVPSEQ ID NO: 414 LKVAQHLAYPVPDV SEQ ID NO: 415 GLKVAQHLAYPVPDSEQ ID NO: 416 GLKVAQHLAYPVPDV SEQ ID NO: 417 PGLKVAQHLAYPVPDSEQ ID NO: 418 ELGFQPGLKVAQHLA SEQ ID NO: 419 LGFQPGLKVAQHLAYSEQ ID NO: 420 GFQPGLKVAQHLAYP SEQ ID NO: 421 QPGLKVAQHLAYPVPSEQ ID NO: 422 FQPGLKVAQHLAYPV SEQ ID NO: 423 LKVAQHLAYPVPDVPSEQ ID NO: 424 FQPGLKVAQHLAYPVP SEQ ID NO: 425 GFQPGLKVAQHLAYPVSEQ ID NO: 426 GLKVAQHLAYPVPDVP SEQ ID NO: 427 PGLKVAQHLAYPVPDVSEQ ID NO: 428 QPGLKVAQHLAYPVPD SEQ ID NO: 429 ELGFQPGLKVAQHLAYSEQ ID NO: 430 LGFQPGLKVAQHLAYP SEQ ID NO: 431 GFQPGLKVAQHLAYPVPSEQ ID NO: 432 FQPGLKVAQHLAYPVPD SEQ ID NO: 433 QPGLKVAQHLAYPVPDVSEQ ID NO: 434 PGLKVAQHLAYPVPDVP SEQ ID NO: 435 LGFQPGLKVAQHLAYPVSEQ ID NO: 436 ELGFQPGLKVAQHLAYP SEQ ID NO: 437 ELGFQPGLKVAQHLAYPVSEQ ID NO: 438 QPGLKVAQHLAYPVPDVP SEQ ID NO: 439 LGFQPGLKVAQHLAYPVPSEQ ID NO: 440 FQPGLKVAQHLAYPVPDV SEQ ID NO: 441 GFQPGLKVAQHLAYPVPDSEQ ID NO: 442 FQPGLKVAQHLAYPVPDVP SEQ ID NO: 443 LGFQPGLKVAQHLAYPVPDSEQ ID NO: 444 GFQPGLKVAQHLAYPVPDV SEQ ID NO: 445 ELGFQPGLKVAQHLAYPVPSEQ ID NO: 446 LGFQPGLKVAQHLAYPVPDV SEQ ID NO: 447 ELGFQPGLKVAQHLAYPVPDSEQ ID NO: 448 GFQPGLKVAQHLAYPVPDVP SEQ ID NO: 449 ELGFQPGLKVAQHLAYPVPDVSEQ ID NO: 450 LGFQPGLKVAQHLAYPVPDVP SEQ ID NO: 451

The peptides that mimic the helices and functional fragments thereof areadministered in formulations and by routes well understood. A variety ofmethods for introducing such substances are known, typically, byinjection, aerosol administration, suppository, and, with proper design,oral administration. This general statement is true as well with respectto providing expression systems for peptides represented by the helixmimics.

In addition to the peptides or other compounds of the invention,combination therapies including known HCV inhibitors can be utilized inthe present invention. For example, it may be desirable to administerboth a peptide or peptides of the invention in combination withinterferon to a subject infected with HCV. Other drugs or compoundsknown in the art to be effective against HCV, can also be used.

In one embodiment, the invention provides a method of screeningcompounds, to identify those that selectively inhibit the binding of HCVnonstructural proteins (for example, NS4B or NS5A) and cellularmembranes. Methods known to those of skill in the art, can be readilyadapted to detect interference with the binding of these components. Themethod of screening may involve high-throughput techniques. For example,to screen for compounds that selectively inhibit the binding of HCVnonstructural proteins and cellular membranes, a synthetic reaction mix,a viral fragment or component, or a preparation of any thereof,comprising an HCV nonstructural protein and a labeled substrate orligand of such polypeptide is incubated in the absence or the presenceof a candidate molecule that may inhibit the binding of the HCVnonstructural proteins and the cellular membrane. The ability of thecandidate molecule to inhibit the binding of the HCV nonstructuralprotein and the cellular membrane is reflected in decreased binding ofthe labeled ligand or decreased production of product from suchsubstrate.

In another aspect, the screening can be performed by adding thecandidate compound to intact cells that have been infected by HCV, orthat contain an HCV replicon, and then examining the component ofinterest to demonstrate the effect on this component, or the effect onviral or replicon replication. An exemplary cell-based in vitro assayfor this purpose is disclosed in PCT International Publication WO02/18369. Alternatively, the screening can be performed by adding thetest agent to in vitro translation reactions and then proceeding withthe established analysis. As another alternative, purified or partiallypurified components which have been determined to interact with oneanother by the methods described above can be placed under conditions inwhich the interaction between them would normally occur, with andwithout the addition of the candidate compound, and the procedurespreviously established to analyze the interaction can be used to assessthe impact of the candidate compound. However their anti-HCV activity isinitially assayed, peptide or other inhibitors of the present inventionshould cause inhibition of infection, replication, or pathogenesis ofHepatitis C Virus in vitro or in vivo when introduced into a host cellcontaining the virus, and exhibit an IC₅₀ in the range of from about0.0001 nM to 100 μM in an in vitro assay for at least one step ininfection, replication, or pathogenesis of HCV, more preferably fromabout 0.0001 nM to 75 μM, more preferably from about 0.0001 nM to 50 μM,more preferably from about 0.0001 nM to 25 μM, more preferably fromabout 0.0001 nM to 10 μM, and even more preferably from about 0.0001 nMto 1 μM.

In another embodiment of the invention, the method of screening may beby phage display. A method of obtaining selective ligands that bind achosen target is to select from a library of proteins or peptidesdisplayed on phage. In order to obtain a novel binding protein against achosen target, such as an amphipathic helix region of an HCV component,DNA molecules, each encoding a protein or peptide fragment thereof, anda structural signal calling for the display of the protein on the outersurface of a chosen genetic package (bacterial cell, bacterial spore orphage) are introduced into a genetic package. The protein is expressedand the potential binding domain is displayed on the outer surface ofthe package. The package is then exposed to the target. If the geneticpackage binds to the target, then it is confirmed that the correspondingbinding domain is indeed displayed by the genetic package. Packageswhich display the binding domain (and thereby bind the target) areseparated from those which do not. For example, in the presentinvention, the target may be the amphipathic helix or a mutatedamphipathic helix. Potential peptides, which may then be used asanti-HCV agents, are screened by determination of which will bind to theamphipathic helix or a mutated amphipathic helix. Preferred peptides arethose that not only bind to the amphipathic helix, but in addition,block or inhibit the amphipathic helix from binding to its receptor orbinding site, thereby inhibiting infectivity. Examples of peptidesidentified by phage display are set forth in Table 3.

TABLE 3 Peptides identified by phaae display asligands of the amphipathic helix of NS5A: Sequence IdentificationSequence Number HDSFANATGRFWP SEQ ID NO: 454 QGTSPSRLAVPLASEQ ID NO: 455 ISSKTGMSSEPPS SEQ ID NO: 456 ILSSIDALGSDSH SEQ ID NO: 457LDDRSVPTVISQR SEQ ID NO: 458 YPSKPGNVTPKAP SEQ ID NO: 459 QAQGERALKSEQ ID NO: 460 TDKRASPLTVQAR SEQ ID NO: 461

In all of the embodiments of the invention, the active agent which willinteract, generally, either with the sites on the cytoplasmic membraneto which the amphipathic helix binds or will interact with theamphipathic helix itself, may be derivatized or coupled to additionalcomponents. By “derivatives” of these agents is meant modificationsthereof that do not interfere with their ability to interact with thesites or the helix, but may optionally confer some additional usefulproperty. One particularly useful property is the ability to penetratecell membranes, and preferred among the derivatives or conjugated formsare those coupled to such facilitators. An additional desired coupledcomponent may be a labeling component such as a fluorescent dye, anenzyme, or a radioisotope. One particularly useful label is, forexample, green fluorescent protein in any of its many colors and forms.Green fluorescent protein thus, includes, not only forms of thisfluorescent protein that fluoresce green, but also those that fluorescevarious other colors such as red and blue. These forms of the proteinare commercially available as are recombinant materials for theirproduction.

The compositions of the invention can be administered as conventionalHCV therapeutics. The compositions of the invention may include morethan one ingredient which interrupts the binding of the amphipathichelix to the membranes and more than one peptide of the invention.

The precise formulations and modes of administration of the anti-HCVcompositions of the invention will depend on the nature of the anti-HCVagent, the condition of the subject, and the judgment of thepractitioner. Design of such administration and formulation is routineoptimization generally carried out without difficulty by thepractitioner.

In addition to the assay methods, methods to identify compounds orprotocols against HCV infection, and methods to treat HCV infections asset forth above, the invention provides compositions which are effectiveto elicit immunological responses to HCV in appropriate subjects, suchas humans or other animals subject to HCV infection.

Administration may be performed using conventional methods, typically byinjection. The elicited immunological response is helpful in general HCVprophylaxis.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLES Example 1 Effect of Helix Disruption on Membrane Association

The bottom panels of FIGS. 3A-3C show the structures of the relevantportion of NS5A used in this example. FIG. 3A shows the amino acidsequence and helix formed at positions 4-27 in the NS5A protein ofHepatitis C virus genotype 1a. FIG. 3B shows the deletion of positions7-27, thus deleting the helix, as shown by the brackets. This deletionwas conducted by PCR mutagenesis essentially as described by Glenn, J.S., et al., J Virol (1998) 72:9303-9306 on plasmid pBRTM/HCV 827-3011which encodes the HCV nonstructural proteins as described by Grakoui,A., et al., J. Virol. (1993) 67:1385-1395. This vector encodes the NSproteins under the T7 promoter and encephalomyocarditis internalribosome entry site control elements and directs the synthesis andprocessing of proteins NS3, NS4A, NS4B, NS5A and NS5B. FIG. 3C shows amutant which was obtained using PCR mutagenesis as described above usinga primer

(SEQ ID NO: 452) (5′-TCCGGCTCCTGGCTAAGGGAC GA CTGGGACTGG GA ATGCGAGGTGCT-GAGCGAC GA TAAGACC-3′)in which codons isoleucine-8, isoleucine-12, and phenylalanine-19 ofNS5A were changed to encode aspartate, glutamate, and aspartate,respectively. This results in disruption of the hydrophobic region ofthe helix.

Each of these plasmids was expressed and distribution of the NS5Aprotein, which is produced by the plasmids in cells, was observed. Aliver derived cell line, Huh-7, was first infected with recombinantvaccinia virus encoding T7-RNA polymerase and then transfected withpBRTM/HCV 827-3001, or by this plasmid modified as described in FIGS. 3Band 3C.

After incubation to effect protein production, the cells were fixed withformaldehyde and immunostained with a monoclonal antibody against NS5Aand a Texas Red-labeled donkey anti-mouse secondary antibody essentiallyas described by Glenn, J. S., et al., J. Virol. (1990) 64:3104-3107. Asshown in the upper panel of FIG. 3A, perinuclear punctate vesicularstaining suggestive of a Golgi-like intracellular distribution patternwas readily observed, as was, occasionally, a somewhat reticulinchicken-wire-like staining pattern, characteristic of the ER. Bothpatterns have been reported previously when NS5A is expressed eitheralone or in combination with other HCV NS proteins using a variety ofexpression systems. See, for example, Kim, J. E., et al., Arch. Virol.(1999) 144:329-343; Huang, Y., et al., Biochem. Biophys. Res. Commun.(2001) 281:732-740.

However, when the construct of FIG. 3B, containing a deleted portion ofNS5A was expressed, as shown in the upper panel of FIG. 3B, cytoplasmicmembrane localization was abolished and a nuclear localization patternwas obtained. A cryptic nuclear localization signal in the NS5AC-terminal domain has previously been reported by Ide, Y., et al., Gene(1996) 182:203-211.

When the construct described in FIG. 3C, which disrupts the hydrophobicface of the helix, is expressed in these cells, again, as shown in theupper panel of FIG. 3C, the cytoplasmic membrane localization patternwas lost and localization to the nucleus was observed.

FIGS. 3A-3C show these results at two magnification levels. Theuppermost panel in each case shows results for a single cell and theintermediate panel shows results for a number of cells. As shown in theintermediate panels in FIGS. 3B and 3C, occasionally distribution of thedye throughout the cell was observed rather than localization to thenucleus. However, even in these cases, no specific localization to thecytoplasmic membrane could be detected.

Example 2 Intracellular Location of GFP-Labeled Helix

Three constructs were made. First, as a control, the HCV encodingsequences of pBRTM/HCV 827-3011 were replaced with the coding sequencefor jellyfish Aequorea victoria green fluorescent protein (GFP) using aPCR cloned insert from plasmid C109 which contains E-GFP (Clontech), toobtain “T7-GFP.” A fragment of the NS5A gene encoding the amphipathichelix was fused into frame with the sequence encoding the N-terminus ofGFP in T7-GFP by creating a common PstI site using PCR mutagenesis. Thispermits junction of the first 31 amino acids of NS5A to the 5′ side ofserine-2 of the GFP-encoding sequence in T7-GFP. The resulting vectorwas labeled “T7-5AGFP.” A similar vector, designated “T7-5ANHGFP” wasconstructed in a manner similar to “T7-5AGFP” except that thecorresponding segment from the mutated form of NS5A set forth in FIG. 3Cwas employed in place of the wildtype helix. Thus, “T7-5ANHGFP” issimilar to “T7-5AGFP” except that in the NS5A helix, Ile at position 8is converted to Asp, Ile at position 12 is converted to Glu, and Phe atposition 19 is converted to Asp.

These constructs were used to effect expression of GFP or of the GFPfusions in Huh-7 cells as described in Example 1 and the distribution offluorescence was evaluated with the results as shown in FIGS. 4A-4C. Asshown in FIG. 4A, cells expressing GFP alone show a wide diffuse patternincluding concentration in the nucleus. Cells expressing 5AGFP showdistribution of fluorescence similar to that of the NS5A protein itselfwith a Golgi-like intracellular distribution pattern and ER staining.Cells expressing 5ANHGFP failed to show this pattern but instead mimicthe distribution pattern of GFP.

Example 3 Effect of Helix Disruption on HCV RNA Replication

Comparative high efficiency, second generation, bicistronic, subgenomicRNA replicons of HCV described by Blight, K. J., et al., Science (2001)290:1972-1974 were constructed with a neomycin-resistance gene. Similarconstructs were made with wildtype NS5A and with NS5A altered in theamphipathic helix as described in Examples 1 and 2. Diagrams of theseconstructs are shown in the upper panels of FIGS. 5A and 5B.

The construct containing the wildtype NS5A, Bart79I, was made by PCRmutagenesis of HCVrep1bBartMan/AvaII (Blight, supra) such thatnucleotide 5336 was changed from a G to T resulting in a change in NS5Acodon 1179 from serine to isoleucine. This mutation results in adramatic increase in replication efficiency of the HCV subgenomicreplicon. Bart5×79I (containing the modified helix) was made by PCRmutagenesis of Bart79I using a primer

(SEQ ID NO: 453) (5′-G A TTGGGATTGG GA ATGCACGGTGTTGACTGAT GA CAAGACCTGG-3′)in which codons valine-8, isoleucine-12, and phenylalanine-19 of NS5Awere changed to encode aspartate, glutamate, and aspartate,respectively. All mutations were confirmed by DNA sequencing.

The RNA replication efficiency was then assayed by ability to establishG418-resistant colonies after transfection of Huh-7 cells as follows:Replicon RNA's were prepared by in vitro transcription with T7-RNApolymerase of ScaI-linearized plasmids (Bart79I or Bart5×79I) followedby DNase treatment and purification. Replicons were then electroporatedinto Huh-7 cells and neomycin-resistant colonies selected by inclusionof 1 mg/ml G418 in the culture media. Methods were essentially asdescribed by Blight (supra). Colonies were detected by staining withcresyl violet. Multiple independent preparations of replicons andreplication assays yielded similar results.

As shown in FIG. 5A, lower panel, the replicon with wildtype NS5AN-terminus gave rise to numerous colonies while disrupting theamphipathic nature of the N-terminal helix results in a dramaticinhibition of HCV genome replication as shown in the lower panel of FIG.5B.

There was no decreased transfection efficiency or any decrease in theability to establish colonies when control experiments using plasmidsencoding drug resistance marker along with only the wildtype or mutantNS5A proteins were used in place of the replicons above, thusestablishing that the results were not due to an increased cytotoxicityassociated with mutant NS5A.

Example 4 Floatation Assay

In this membrane floatation assay, the ability of the NS5A amphipathichelix to bind to a preparation of microsomal membranes was tested. NS5Aproteins, both the NS5A wildtype or mutant amphipathic helix (describedin Example 1), were in vitro translated and [³⁵S]-labeled using thePromega TNT reticulocyte lysate kit. Plasmids pC5A or pC5ANH were usedas transcription templates. Aliquots of the reactions were combined witha membrane fraction derived from Huh-7 cells, with or without syntheticpeptides, and overlayed with a 5-40% OptiPrep step gradient. Aftercentrifugation for 4 hrs at 40,000×g in a SW60 rotor, 500 ml fractionswere collected from the top and the proteins in each gradient fractionwere precipitated and analyzed by SDS-PAGE. The percentage of protein“floating” to the top of the gradient with membranes (fraction 2 fromthe top) was then quantified using a Molecular Dynamics phosphorimager.

FIGS. 6A and 6B show a comparison of the results for wildtype and mutantforms. The numbers correspond to Optiprep gradient (5-40%) fractionnumber. Non-membrane associated proteins remain at the bottom (rightside) of the gradient, whereas membranes—and associated proteins—“float”towards less dense gradient fractions present at the top (left side). Asshown, in the reaction mixture containing wildtype, the synthesized NS5Afloated toward the top of the gradient coupled with the membrane. Thiswas not found in the case of the mutant form. In both cases, there was adegree of non-specific binding associated with fractions that settle tothe bottom of the gradient. An advantage of this method is eliminationof the artifacts caused by non-specific binding, as illustrated by thelabeling of high density fractions in both mixtures.

FIG. 6C shows the percentage of NS5A wildtype or mutant protein thatfloat with the membrane fractions as a percentage of the totalsynthesized. As seen, 25% of the wildtype, but only 0.8% of the mutant,was associated with the membrane.

FIGS. 7A and 7B show the results of a similar floatation experiment inthe presence and absence of a test peptide that is a competitiveinhibitor of the NS5A helix. This peptide contains amino acids 1-27 ofNS5A with a C-terminal “flag” DYKD. The assay described in the precedingparagraph was repeated in the presence and absence of this peptide. Asshown in FIG. 7A, the percentage of NS5A floated in the absence ofpeptide is arbitrarily normalized to 100%; in comparison with this, thepresence of the test peptide lowered the percentage to about 21%.

Similarly, the assay was modified by using, in place of NS5A, a fusionprotein consisting of the amphipathic helix of NS5A with greenfluorescent protein. As can be seen in FIG. 7B, the amount of labeledNS5A floated with the membrane was set in the absence of test peptide at100%. In comparison, the presence of the test peptide lowered thepercentage floated to about 21%.

Moreover, as shown in FIG. 6C, a synthetic peptide designed to mimic thewild type AH not only competitively inhibited membrane association ofNS5A, but did so in a dose-dependent manner. The maximal extent ofinhibition achieved pharmacologically was comparable to that obtained bygenetic mutation of the AH. In addition, the synthetic peptide appearsequally effective against NS5A derived from different genotypes (datanot shown), including those most refractory to current therapies. Thisconvenient membrane floatation assay has also proven well-suited forcurrent efforts focused on studying the mechanistic details of the AHmembrane-targeting domain, and ideal for guiding ongoing development ofpeptidomimetic compounds designed to resemble key elements, or bind tospecific features, of the AH. Such compounds represent an excitingpotential addition to current anti-HCV combination therapy regimens.

FIG. 8 shows Pharmacologic inhibition of NS5A membrane association.Huh-7 cells-derived membranes were treated with PEP1 (a peptidemimicking the wild type amphipathic helix of NS5A), PEP2 (a controlpeptide) or mock-treated with water. PEP1 (a peptide that corresponds tothe wild type N-terminal amphipathic helix of NS5A (amino acids 1-26)with a C-terminal FLAG tag) and PEP2 (which is identical to PEP1 exceptamino acids isoleucine-8, isoleucine-12, and phenylalanine-19 of NS5Awere changed to aspartate, glutamate, and aspartate, respectively) weresynthesized by AnaSpec Inc. In-vitro translated NS5A (or NS5ANH) wasincubated with membranes and various concentrations of peptides or waterprior to analysis by membrane floatation assays set forth above. Theamount of NS5A floating with no peptide treatment typically is ˜30% ofthe total in-vitro translated NS5A. For comparison between treatmentconditions, results were normalized to the no-treatment control. Thepercent of NS5A (or NS5ANH) floating with the membranes under theindicated conditions was determined as in FIG. 6A and expressed relativeto mock-treated control. Error bars represent standard error of themean.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

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1. A composition comprising an isolated peptide of 10 to 29 amino acidsin length, wherein the isolated peptide comprises the sequence:LRDIWDWICEVLSDFKTWLKA; (SEQ ID NO: 2) WLRDVWDWICTVLTDFKTWLQSKL;(SEQ ID NO: 5) WLRDVWDWVCTILTDFKNWLTSKL; (SEQ ID NO: 6)WLRDIWEWVLSILTDFKNWLSAKL; (SEQ ID NO: 7) WLRIIWDWVCSVVSDFKTWLSAKI;(SEQ ID NO: 8) WLRTIWDWVCSVLADFKAWLSAKI; (SEQ ID NO: 9)WLHDIWDWVCIVLSDFKTWLSAKI; (SEQ ID NO: 10) WLWDVWDWVLHVLSDFKTCLKAKF;(SEQ ID NO: 11) WLYDIVNWVCTVLADFKLWLGAKI; (SEQ ID NO: 12)WLRDIWDWVCTVLSDFRVWLKSKL; (SEQ ID NO: 13) SGSWLRDVWDWICTVLTDFKTWLQSKL;(SEQ ID NO: 14) LRDVWDWICTVLTDFKT; (SEQ ID NO: 463) LRDVWDWICT;(SEQ ID NO: 464) DVWDWICTVLTD; (SEQ ID NO: 465) and SWLRDVWDWIC;(SEQ ID NO: 466) LRDVWDWICVLTD; (SEQ ID NO: 471)SGSWLRDVWDWICTVATDFKTWLQSKL; (SEQ ID NO: 472)SGSWLRDVWDWICTVKTDFKTWLQSKL; (SEQ ID NO: 473)SGSWLRDVWDWICTVLADFKTWLQSKL; (SEQ ID NO: 474)SGSWLRDVWDWICTVLTAFKTWLQSKL; (SEQ ID NO: 475)SGSWLRDVWDWICTVLTDAKTWLQSKL; (SEQ ID NO: 476)SGSWLRDVWDWICTVLTDFKTALQSKL; (SEQ ID NO: 478) orSGSWLRDVWDWICTVLTDFKTWKQSKL, (SEQ ID NO: 479)

wherein the peptide comprises a non-natural amino acid, wherein thepeptide retains the ability to form an amphipathic helix, and whereinthe isolated peptide inhibits infection by and/or replication ofHepatitis C Virus in vitro.
 2. The composition of claim 1, wherein thenon-natural amino acid is a D-amino acid.
 3. The composition of claim 1,wherein the peptide is coupled to a membrane-penetrating facilitator. 4.The composition of claim 1, wherein the peptide comprises a non-peptideisosteric linkage.
 5. The composition of claim 1, wherein the peptide isbound to a solid support.
 6. The composition of claim 1, wherein thepeptide comprises a detectable label.
 7. The composition of claim 1,wherein the peptide comprises the sequence: LRDIWDWICEVLSDFKTWLKA (SEQID NO:2).
 8. The composition of claim 1, wherein the peptide comprisesthe sequence: WLRDVWDWICTVLTDFKTWLQSKL (SEQ ID NO:5).
 9. The compositionof claim 1, wherein the peptide comprises the sequence:WLRDVWDWVCTILTDFKNWLTSKL (SEQ ID NO:6).
 10. The composition of claim 1,wherein the peptide comprises the sequence: WLRDIWEWVLSILTDFKNWLSAKL(SEQ ID NO:7).
 11. The composition of claim 1, wherein the peptidecomprises the sequence: WLRIIWDWVCSVVSDFKTWLSAKI (SEQ ID NO:8).
 12. Thecomposition of claim 1, wherein the peptide comprises the sequence:WLRTIWDWVCSVLADFKAWLSAKI (SEQ ID NO:9).
 13. The composition of claim 1,wherein the peptide comprises the sequence: WLHDIWDWVCIVLSDFKTWLSAKI(SEQ ID NO:10).
 14. The composition of claim 1, wherein the peptidecomprises the sequence: WLWDVWDWVLHVLSDFKTCLKAKF (SEQ ID NO:11).
 15. Thecomposition of claim 1, wherein the peptide comprises the sequence:WLYDIVNWVCTVLADFKLWLGAKI (SEQ ID NO:12).
 16. The composition of claim 1,wherein the peptide comprises the sequence: WLRDIWDWVCTVLSDFRVWLKSKL(SEQ ID NO:13).
 17. The isolated peptide of claim 1, wherein the peptidecomprises the sequence: SGSWLRDVWDWICTVLTDFKTWLQSKL (SEQ ID NO: 14). 18.The composition of claim 1, wherein the peptide comprises the sequence:LRDVWDWICTVLTDFKT (SEQ ID NO:463).
 19. The composition of claim 1,wherein the peptide comprises the sequence: LRDVWDWICT (SEQ ID NO:464).20. The composition of claim 1, wherein the peptide comprises thesequence: DVWDWICTVLTD (SEQ ID NO:465).
 21. The composition of claim 1,wherein the peptide comprises the sequence: SWLRDVWDWIC (SEQ ID NO:466).22. The composition of claim 1, wherein the peptide comprises thesequence: LRDVWDWICTVLTD (SEQ ID NO:471).
 23. The composition of claim1, wherein the peptide comprises the sequence:SGSWLRDVWDWICTVATDFKTWLQSKL (SEQ ID NO:472).
 24. The composition ofclaim 1, wherein the peptide comprises the sequence:SGSWLRDVWDWICTVKTDFKTWLQSKL (SEQ ID NO:473).
 25. The composition ofclaim 1, wherein the peptide comprises the sequence:SGSWLRDVWDWICTVLADFKTWLQSKL (SEQ ID NO:474).
 26. The composition ofclaim 1, wherein the peptide comprises the sequence:SGSWLRDVWDWICTVLTAFKTWLQSKL (SEQ ID NO:475).
 27. The composition ofclaim 1, wherein the peptide comprises the sequence:SGSWLRDVWDWICTVLTDAKTWLQSKL (SEQ ID NO:476).
 28. The composition ofclaim 1, wherein the peptide comprises the sequence:SGSWLRDVWDWICTVLTDFKTALQSKL (SEQ ID NO:478).
 29. The composition ofclaim 1, wherein the peptide comprises the sequence:SGSWLRDVWDWICTVLTDFKTWKQSKL (SEQ ID NO:479).